Strains of agrobacterium modified to increase plant transformation frequency

ABSTRACT

Agrobacterium  strains that harbor transformation-enhancing genes on a plasmid capable of replication independently of the  Agrobacterium  chromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these  Agrobacterium  strains are provided. Additionally,  Agrobacterium  strains that are deficient in DNA recombination functions that result in instability or rearrangement of plant transformation binary vectors, and that harbor transformation-enhancing genes on a plasmid capable of replication independently of the  Agrobacterium  chromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these strains, are also provided. Further included are  Agrobacterium  strains that harbor transformation-enhancing genes integrated into the  Agrobacterium  chromosome at a locus that does not interfere with or otherwise compromise the normal growth and plant transformation ability of the  Agrobacterium  cells, and uses for these  Agrobacterium  strains. Plants made using these  Agrobacterium  strains are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/812,469 filed on Mar. 21, 2013, which is a national phase entry under35 U.S.C. §371 of International Patent Application PCT/US2011/046028,filed Jul. 29, 2011, designating the United States of America andpublished in English as International Patent Publication WO 2012/016222on Jun. 21, 2012, which claims the benefit under Article 8 of the PatentCooperation Treaty and under 35 U.S.C. §119(e) to U.S. ProvisionalPatent Application Ser. No. 61/368,965, filed Jul. 29, 2010, thedisclosure of each of which is hereby incorporated herein by thisreference in its entirety.

TECHNICAL FIELD

The present invention relates to Agrobacterium strains that harbortransformation-enhancing genes on a plasmid capable of replicationindependently of the Agrobacterium chromosome, the Ti plasmid, and planttransformation binary vectors, and uses for these Agrobacterium strains.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)-SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing an ASCII textversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

BACKGROUND

Plant transformation generally encompasses the methodologies requiredand utilized for the introduction of a plant-expressible foreign geneinto plant cells, such that fertile progeny plants may be obtained whichstably maintain and express the foreign gene. Numerous members of themonocotyledonous and dicotyledonous classifications have beentransformed. Transgenic agronomic crops, as well as fruits andvegetables, are of commercial interest. Such crops include but are notlimited to maize, rice, soybeans, canola, sunflower, alfalfa, sorghum,wheat, cotton, peanuts, tomatoes, potatoes, and the like. Despite thedevelopment of plant transformation systems for introducingplant-expressible foreign genes into plant cells, additionalimprovements which allow for increased transformation efficiency aredesirable and provide significant advantages in overcoming operationaldisadvantages when transforming plants with foreign genes.

Several techniques are known for introducing foreign genetic materialinto plant cells, and for obtaining plants that stably maintain andexpress the introduced gene. Such techniques include acceleration ofgenetic material coated onto microparticles directly into cells (e.g.,U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Othertransformation technology includes silicon carbide or WHISKERS™technology. See, e.g., U.S. Pat. No. 5,302,523 and U.S. Pat. No.5,464,765. Electroporation technology has also been used to transformplants. See, e.g., WO 87/06614, U.S. Pat. No. 5,472,869, U.S. Pat. No.5,384,253, WO 92/09696, and WO 93/21335. Additionally, fusion of plantprotoplasts with liposomes containing the DNA to be delivered, directinjection of the DNA, as well as other possible methods, may beemployed.

Once the inserted DNA has been integrated into the plant genome, it isusually relatively stable throughout subsequent generations. Thetransformed cells grow inside the plants in the usual manner. They canform germ cells and transmit the transformed trait(s) to progeny plants.Such plants can be grown in the normal manner and may be crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties, for example, the ability to controlthe feeding of plant pest insects.

A number of alternative techniques can also be used for inserting DNAinto a host plant cell. Those techniques include, but are not limitedto, transformation with T-DNA delivered by Agrobacterium tumefaciens orAgrobacterium rhizogenes as the transformation agent. Plants may betransformed using Agrobacterium technology, as described, for example,in U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, European PatentApplication No. 0131624B1, European Patent Application No. 120516,European Patent Application No. 159418B1, European Patent ApplicationNo. 176112, U.S. Pat. No. 5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat.No. 5,464,763, U.S. Pat. No. 4,940,838, U.S. Pat. No. 4,693,976,European Patent Application No. 116718, European Patent Application No.290799, European Patent Application No. 320500, European PatentApplication No. 604662, European Patent Application No. 627752, EuropeanPatent Application No. 0267159, European Patent Application No. 0292435,U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No.4,762,785, U.S. Pat. No. 5,004,863, and U.S. Pat. No. 5,159,135. The useof T-DNA-containing vectors for the transformation of plant cells hasbeen intensively researched and sufficiently described in EuropeanPatent Application 120516; An et al. (1985, EMBO J. 4:277-284), Fraleyet al. (1986, Crit. Rev. Plant Sci. 4:1-46), and Lee and Gelvin (2008,Plant Physiol. 146: 325-332), and is well established in the field.

The biology of T-DNA transfer from Agrobacterium to plant cells isknown. See, e.g., Gelvin (2003) Microbiol. Molec. Biol. Rev. 67:16-37;and Gelvin (2009) Plant Physiol. 150:1665-1676. At minimum, at least aT-DNA right border repeat, but often both the right border repeat andthe left border repeat of the Ti or Ri plasmid will be joined as theflanking region of the genes desired to be inserted into the plant cell.The left and right T-DNA border repeats are crucial cis-acting sequencesrequired for T-DNA transfer. Various trans-acting components are encodedwithin the total Agrobacterium genome. Primary amongst these are theproteins encoded by the vir genes, which are normally found as a seriesof operons on the Ti or Ri plasmids. Various Ti and Ri plasmids differsomewhat in the complement of vir genes, with, for example, virF notalways being present. Proteins encoded by vir genes perform manydifferent functions, including recognition and signaling of plantcell/bacteria interaction, induction of vir gene transcription,formation of a Type IV secretion channel, recognition of T-DNA borderrepeats, formation of T-strands, transfer of T-strands to the plantcell, import of the T-strands into the plant cell nucleus, andintegration of T-strands into the plant nuclear chromosome, to name buta few. See, e.g., Tzfira and Citovsky (2006) Curr. Opin. Biotechnol.17:147-154.

If Agrobacterium strains are used for transformation, the DNA to beinserted into the plant cell can be cloned into special plasmids, forexample, either into an intermediate (shuttle) vector or into a binaryvector. Intermediate vectors are not capable of independent replicationin Agrobacterium cells, but can be manipulated and replicated in commonEscherichia coli molecular cloning strains. Such intermediate vectorscomprise sequences are commonly framed by the right and left T-DNAborder repeat regions, that may include a selectable marker genefunctional for the selection of transformed plant cells, a cloninglinker, a cloning polylinker, or other sequence which can function as anintroduction site for genes destined for plant cell transformation.Cloning and manipulation of genes desired to be transferred to plantscan thus be easily performed by standard methodologies in E. coli, usingthe shuttle vector as a cloning vector. The finally manipulated shuttlevector can subsequently be introduced into Agrobacterium planttransformation strains for further work. The intermediate shuttle vectorcan be transferred into Agrobacterium by means of a helper plasmid (viabacterial conjugation), by electroporation, by chemically mediateddirect DNA transformation, or by other known methodologies. Shuttlevectors can be integrated into the Ti or Ri plasmid or derivativesthereof by homologous recombination owing to sequences that arehomologous between the Ti or Ri plasmid, or derivatives thereof, and theintermediate plasmid. This homologous recombination (i.e., plasmidintegration) event thereby provides a means of stably maintaining thealtered shuttle vector in Agrobacterium, with an origin of replicationand other plasmid maintenance functions provided by the Ti or Ri plasmidportion of the co-integrant plasmid. The Ti or Ri plasmid also comprisesthe vir regions comprising vir genes necessary for the transfer of theT-DNA. The plasmid carrying the vir region is commonly a mutated Ti orRi plasmid (helper plasmid) from which the T-DNA region, including theright and left T-DNA border repeats, have been deleted. Such pTi-derivedplasmids, having functional vir genes and lacking all or substantiallyall of the T-region and associated elements are descriptively referredto herein as helper plasmids.

The superbinary system is a specialized example of the shuttlevector/homologous recombination system (reviewed by Komari et al. (2006)in Methods in Molecular Biology (K. Wang, ed.) No. 343: AgrobacteriumProtocols (2^(nd) Edition, Vol. 1) HUMANA PRESS Inc., Totowa, N.J., pp.15-41; and Komori et al. (2007) Plant Physiol. 145:1155-1160). TheAgrobacterium tumefaciens host strain employed with the superbinarysystem is LBA4404(pSB1). Strain LBA4404(pSB1) harbors two independentlyreplicating plasmids, pAL4404 and pSB1. pAL4404 is a Ti-plasmid-derivedhelper plasmid which contains an intact set of vir genes (from Tiplasmid pTiACH5), but which has no T-DNA region (and thus no T-DNA leftand right border repeat sequences). Plasmid pSB1 supplies an additionalpartial set of vir genes derived from pTiBo542; this partial vir geneset includes the virB operon and the virC operon, as well as genes virGand virD1. One example of a shuttle vector used in the superbinarysystem is pSB11, which contains a cloning polylinker that serves as anintroduction site for genes destined for plant cell transformation,flanked by right and left T-DNA border repeat regions. Shuttle vectorpSB11 is not capable of independent replication in Agrobacterium, but isstably maintained as a co-integrant plasmid when integrated into pSB1 bymeans of homologous recombination between common sequences present onpSB1 and pSB11. Thus, the fully modified T-DNA region introduced intoLBA4404(pSB1) on a modified pSB11 vector is productively acted upon andtransferred into plant cells by Vir proteins derived from two differentAgrobacterium Ti plasmid sources (pTiACH5 and pTiBo542). The superbinarysystem has proven to be particularly useful in transformation of monocotplant species. See Hiei et al. (1994) Plant J. (6:271-282); and Ishidaet al. (1996) Nat. Biotechnol. 14:745-750.

In addition to the vir genes harbored by Agrobacterium Ti plasmids,other, chromosomally borne virulence controlling genes (termed chvgenes) are known to control certain aspects of the interactions ofAgrobacterium cells and plant cells, and thus affect the overall planttransformation frequency (Pan et al. (1995) Molec. Microbiol.17:259-269). Several of the chromosomally borne genes required forvirulence and attachment are grouped together in a chromosomal locusspanning 29 kilobases (Matthysse et al. (2000) Biochim. Biophys. Acta1490:208-212).

Regardless of the particular plasmid system employed, the Agrobacteriumcells so transformed are used for the transformation of plant cells.Plant explants (for example, pieces of leaf, segments of stalk, roots,but also protoplasts or suspension-cultivated cells) can advantageouslybe cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenesfor the transfer of the DNA into the plant cell. Whole plants may thenbe regenerated from the infected plant material following placement insuitable growth conditions and culture medium, which may containantibiotics or herbicides for selection of the transformed plant cells.The plants so obtained can then be tested for the presence of theinserted DNA.

These techniques for introducing foreign genetic material into plantscan be used to introduce beneficial traits into the plants. For example,billions of dollars are spent each year to control insect pests andadditional billions are lost to the damage they inflict. Syntheticorganic chemical insecticides have been the primary tools used tocontrol insect pests but biological insecticides, such as theinsecticidal proteins derived from Bacillus thuringiensis (Bt), haveplayed an important role in some areas. The ability to produceinsect-resistant plants through the introduction of Bt insecticidalprotein genes has revolutionized modern agriculture and heightened theimportance and value of insecticidal proteins and their genes.

Several Bt proteins have been used to create the insect-resistanttransgenic plants that have been successfully developed and in manycases registered and commercialized. These include Cry1Ab, Cry1Ca,Cry1Fa, and Cry3Bb in corn, Cry1Ac and Cry2Ab in cotton, and Cry3A inpotato.

The commercial products expressing Bt proteins express a single proteinexcept in cases where the combined insecticidal spectrum of two proteinsis desired (e.g., Cry1Ab and Cry3Bb in corn combined to provideresistance to lepidopteran pests and rootworm, respectively) or wherethe independent action of the proteins makes them useful as a tool fordelaying the development of resistance in susceptible insect populations(e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistancemanagement for tobacco budworm).

That is, some of the qualities of insect-resistant transgenic plantsthat have led to rapid and widespread adoption of this technology alsogive rise to the concern that pest populations will develop resistanceto the insecticidal proteins produced by these plants. Severalstrategies have been suggested for preserving the utility of Bt-basedinsect resistance traits which include deploying proteins at a high dosein combination with a refuge, and alternation with, or co-deployment of,different toxins (McGaughey et al. 1998, Nature Biotechnol. 16:144-146).

If Bt proteins are selected for use in combination, they need to exerttheir insecticidal effect independently so that resistance developed toone protein does not confer resistance to the second protein (i.e.,there is not cross resistance to the proteins). A robust assessment ofcross-resistance is typically made using populations of a pest speciesnormally sensitive to the insecticidal protein that has been selectedfor resistance to the insecticidal proteins. If, for example, a pestpopulation selected for resistance to “Protein A” is sensitive to“Protein B,” we would conclude that there is not cross resistance andthat a combination of Protein A and Protein B would be effective indelaying resistance to Protein A alone.

In the absence of resistant insect populations, assessments can be madebased on other characteristics presumed to be related to mechanism ofaction and cross-resistance potential. The utility of receptor-mediatedbinding in identifying insecticidal proteins likely to not exhibit crossresistance has been suggested (U.S. Pat. No. 6,855,873). The keypredictor of lack of cross resistance integral to this approach is thatthe insecticidal proteins do not compete for receptors in a sensitiveinsect species.

In the event that two Bt Cry toxins compete for the same receptor, thenif that receptor mutates in that insect so that one of the toxins nolonger binds to that receptor and thus is no longer insecticidal againstthe insect, it might also be the case that the insect will also beresistant to the second toxin (which competitively bound to the samereceptor). However, if two toxins bind to two different receptors, thiscould be an indication that the insect would not be simultaneouslyresistant to those two toxins.

Cry1Fa is useful in controlling many lepidopteran pests speciesincluding the European corn borer (ECB; Ostrinia nubilalis (Hubner)) andthe fall armyworm (FAW; Spodoptera frugiperda), and is active againstthe sugarcane borer (SCB; Diatraea saccharalis).

The Cry1Fa protein, as produced in corn plants containing event TC1507,is responsible for an industry-leading insect resistance trait for FAWcontrol. Cry1Fa is further deployed in the HERCULEX®, SMARTSTAX™, andWIDESTRIKE™ products.

The ability to conduct (competitive or homologous) receptor bindingstudies using Cry1Fa protein is limited because the most commontechnique available for labeling proteins for detection in receptorbinding assays inactivates the insecticidal activity of the Cry1Faprotein.

Cry1Ab and Cry1Fa are insecticidal proteins currently used (separately)in transgenic corn to protect plants from a variety of insect pests. Akey pest of corn that these proteins provide protection from is theEuropean corn borer (ECB). U.S. Patent Application No. 2008/0311096relates in part to the use of Cry1Ab to control a Cry1F-resistant ECBpopulation.

This application describes strains of Agrobacterium tumefaciens thathave been modified to increase plant transformation frequency. The useof these strains provides novel plant transformation systems for theintroduction of plant-expressible foreign genes into plant cells. Inaddition, these strains provide additional improvements which allow forincreased transformation efficiency and provide significant advantagesin overcoming operational disadvantage when transforming plants withforeign genes.

SUMMARY OF THE INVENTION

Agrobacterium strains that harbor transformation-enhancing genes on aplasmid capable of replication independently of the Agrobacteriumchromosome, the Ti plasmid, and plant transformation binary vectors andmethods for their use are described herein. The Agrobacterium strainsare deficient in DNA recombination functions that result in instabilityor rearrangement of plant transformation binary vectors, and harbortransformation-enhancing genes on a plasmid capable of replicationindependently of the Agrobacterium chromosome, the Ti plasmid, and planttransformation binary vectors. Additional Agrobacterium strains thatharbor transformation-enhancing genes integrated into the Agrobacteriumchromosome at a locus that does not interfere with or otherwisecompromise the normal growth and plant transformation ability of theAgrobacterium cells and their use are also described.

In one embodiment of the methods described herein, a plant istransformed by contacting a cell of the plant with an Agrobacteriumstrain having at least one pTi helper plasmid comprising a 14.8 KpnIfragment of pSB1 and a pTi plasmid having at least one disarmed T-DNAregion, the T-DNA region comprising at least a right T-DNA border andexogenous DNA adjacent to the border, wherein the plasmids havediffering origins of replication relative to each other.

In a further embodiment of the methods described herein, a plant istransformed by contacting a cell of the plant with a bacterium of thegenus Agrobacterium having a 14.8 KpnI VirBCDG fragment of pSB1 and apTi plasmid having at least one disarmed T-DNA region, wherein the 14.8KpnI VirBCDG fragment has been integrated into a neutral integrationsite of a chromosome of the bacterium.

In an additional embodiment of the methods described herein, anAgrobacterium strain includes at least one pTi helper plasmid comprisinga 14.8 KpnI fragment of pSB1 and a pTi plasmid having at least onedisarmed T-DNA region, wherein the plasmids have differing origins ofreplication relative to each other.

In a further embodiment, an Agrobacterium strain withtransformation-enhancing properties includes a 14.8 KpnI VirBCDGfragment isolated from pSB1 and a pTi plasmid having at least onedisarmed T-DNA region.

In another embodiment, a nilA genomic locus of Agrobacterium tumefaciensincludes a polynucleotide sequence that is integrated into the nilAgenomic locus.

In an additional embodiment, an Agrobacterium strain with a 14.8 KpnIVirBCDG fragment of SB1 is integrated into a neutral integration site onthe Agrobacterium chromosome.

In another embodiment an Agrobacterium strain LB4404 includes a 14.8KpnI VirBCDG fragment of pSB1on a pTi helper plasmid and a pTi plasmidhaving at least one disarmed T-DNA region and has exogenous DNA adjacentto at least one Agrobacterium T-DNA border, wherein the plasmids havediffering origins of replication relative to each other.

In a further embodiment an Agrobacterium strain LBA4404 includes atleast one vir gene from a 14.8 KpnI VirBCDG fragment isolated from pSB1integrated into a neutral integration site on the Agrobacteriumchromosome.

In additional embodiments plants are provided that are made according tothe transformation methods described herein.

In yet another embodiment, a fertile transgenic corn plant, or progenythereof, expresses insecticidal amounts of Cry1Ca protein, Cry1Finsecticidal protein, Cry1Ab1 insecticidal protein, andherbicide-tolerant amounts of AAD-1 protein, wherein the Cry1Ca, Cry1F,Cry1Ab1, and AAD1 proteins are collectively expressed from a singlelocus of recombinant DNA stably incorporated in the genome of the plant.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cloning scheme for the construction of plasmid pDAB9292.

FIG. 2 shows a map of plasmid pDOW3719.

FIG. 3 shows a cloning scheme for the construction of plasmid pDAB9698.

FIG. 4 shows maps of binary vector plasmids pDAB101513 and pDAB101514.

FIG. 5 shows a map of binary vector plasmid pDAB101556.

DETAILED DESCRIPTION OF THE INVENTION

Strains of Agrobacterium differ from one another in their ability totransform plant cells. Wild-type, oncogenic Agrobacterium strains areknown for their ability to induce crown galls (tumorous overgrowths) onmany host plants, especially dicot species. This transformation ofnormally growing plant cells into non-self regulated tumor cells comesabout as the result of the transfer of specialized DNA sequences(T-DNA), which encodes plant expressible genes encoding plant hormones,from the tumor-inducing (Ti) plasmid into the plant cells, wherein theyare stably integrated into plant chromosomes. The Ti plasmid from strainBo542 (i.e., pTiBo542) is notable in that, when placed in someAgrobacterium chromosomal backgrounds, it promotes the induction ofespecially large, vigorously growing tumors on some plants (Hood et al.(1986) J. Bacteriol. 168:1291-1301). The genes responsible for this“supervirulence” phenotype reside on pTiBo542 outside the T-DNA regions.Further work found that a plasmid containing a “15.8” kilobase pair(kbp) KpnI fragment derived from pTiBo542 and which contained the entirevirG, virB, and virC operons promoted increased tumor formation bystrain A281, when compared to strains lacking the plasmid (Jin et al.(1987) J. Bacteriol. 169:4417-4425). The virG gene of pTiBo542 isbelieved to be responsible for the supervirulent phenotype ofAgrobacterium strain A281. virG from pTiBo542 causes a 1.7-fold increasein virB expression compared with virG from pTiA6, due to differencesbetween the two genes in the promoter regions, coding sequences, and 3′untranslated regions (Chen et al. (1991) Molec. Gen. Genet.230:302-309). Thus, the virG gene from pTiBo542 can be advantageouslyused to promote higher T-DNA transfer efficiencies, and thus higherplant transformation frequencies, especially when present on a largeKpnI fragment of the pTiBo542 plasmid that also harbors the pTiBo542virB and virC operons.

The complete, annotated sequence of pTiBo542 was submitted to GENBANK asAccession Number DQ058764 on May 12, 2005. Examination of the KpnIrestriction fragment map and gene annotations reveals that the entirevirB operon (which includes the genes virB1, virB2, virB3, virB4, virB5,virB6, virB7, virB8, virB9, virB10, and virB11), the virG gene, the virCoperon (which comprises genes virC1 and virC2) and the part of the virDoperon comprising gene virD1 are isolatable on a KpnI fragmentcomprising 14,815 base pairs (bp). Assumedly, the size of the “15.8 kbp”KpnI fragment referred to in Jin et al. (supra.) was estimated fromagarose gel mobility of the fragment, and that the true size of thereferenced fragment is, in fact, 14.8 kbp. One skilled in the field ofmolecular biology will understand that size estimation of such large DNAfragments by means of agarose gel electrophoresis mobility can differfrom the true fragment size determined by DNA sequence analysis by 1 kbpor more. For ease of description, this fragment derived from pTiBo542will be referred to herein as the 14.8 KpnI VirBCDG fragment.

An embodiment of methods described herein includes uses of thetransformation-enhancing properties encoded on the 14.8 KpnI VirBCDGfragment isolated from pSB1 in Agrobacterium strains harboring at leastone disarmed pTi helper plasmid, wherein the 14.8 KpnI VirBCDG fragmentis borne on a plasmid having a replication origin of an incompatibilitygroup other than IncP to transform a plant. A further embodimentincludes the Agrobacterium strain as described for use in the method. AT-DNA region to be introduced to a plant using this Agrobacterium straincan be borne on a plasmid having a T-DNA region adjacent to at least oneAgrobacterium T-DNA border, the plasmid having a replication origin ofan IncP incompatibility group or an incompatability group that iscompatible with the incompatibility group of the 14.8 KpnI VirBCDGfragment that is borne on a plasmid having a replication origin of anincompatibility group other than IncP. The T-DNA region of this plasmidcan be adjacent right and left Agrobacterium T-DNA borders.

Plasmids are assigned to incompatibility groups (genotypic designation:inc; group designation: Inc) based on sequences contained in theplasmid. The inc determinant typically serves to prevent other plasmidsof the same or related incompatibility group from coexisting in the samehost, and helps maintain a certain copy number of the plasmid within thecell. See, e.g., Fernandez-Lopez, et al. (2006) FEMS Microbiol. Rev.30:942-66; and Adamczyk and Jagura-Burdzy (2003) Acta Biochim. Pol.50:425-53. Two plasmids are incompatible if either is less stable in thepresence of the other than it was by itself. Competition for cellresources can result when two plasmids of the same incompatibility groupare found in the same cell. Whichever plasmid is able to replicatefaster, or provides some other advantage, will be represented to adisproportionate degree among the copies allowed by the incompatibilitysystem. Surprisingly, plasmids can also be incompatible when they bothpossess the same functions for partitioning themselves into daughtercells.

Plasmids typically fall into only one of the many existingincompatibility groups. There are more than 30 known incompatibilitygroups. Plasmids belonging to incompatibility group IncP have beenstudied thoroughly and a large number of plasmids which derive from thisIncP group have been constructed (Schmidhauser et al. (1988)Biotechnology 10:287-332). Exemplary plasmids containing the IncPincompatibility group include: pMP90RK, pRK2013, pRK290, pRK404, andpRK415. These plasmids may be maintained in numerous bacterial speciesincluding E. coli and Agrobacterium tumefaciens. Examples of otherincompatibility groups include, but are not limited to; IncN, IncW,IncL/M, IncT, IncU, IncW, IncY, IncB/O, IncFII, IncII, IncK, IncCom9,IncFI, IncFII, IncFIII, IncHI1, IncHI2, IncX, IncA/C, IncD, IncFIV,IncFV/FO, IncFVI, IncH1 3, IncHII, Inc12, Inch, IncJ, IncV, IncQ, andthe like, including variants thereof, e.g., exhibiting substantialsequence or functional relationship. Table 1 lists several commonlyknown incompatibility groups and provides examples of plasmids whichrepresent these incompatibility groups (this listing of incompatabilitygroups and plasmids is provided by way of example only and is notintended to be limiting on the incompatibility groups and plasmidsuseful with the Agrobacterium strains and methods described herein).

Another embodiment of the methods described herein includes uses oftransformation-enhancing properties encoded on the 14.8 KpnI VirBCDGfragment isolated from pSB1 in Agrobacterium strains having a deficiencyin RecA function, and harboring at least one disarmed pTi helperplasmid, wherein the 14.8 KpnI VirBCDG fragment is borne on a plasmidhaving a replication origin of an incompatibility group other than IncP.A further embodiment includes the Agrobacterium strain as described foruse in the method. A T-DNA region to be introduced to a plant using thisAgrobacterium strain can be borne on a plasmid having a T-DNA regionadjacent to at least one Agrobacterium T-DNA border, the plasmid havinga replication origin of an IncP incompatibility group or anincompatability group that is compatible with the incompatibility groupof the 14.8 KpnI VirBCDG fragment that is borne on a plasmid having areplication origin of an incompatibility group other than IncP.

Yet another embodiment of the methods described herein includes uses ofthe transformation-enhancing properties encoded on the 14.8 KpnI VirBCDGfragment isolated from pSB1, and harboring at least one disarmed pTihelper plasmid, wherein the 14.8 KpnI VirBCDG fragment is integratedinto a chromosomally located neutral integration site of anAgrobacterium strain different from strain C58. A further embodimentincludes the Agrobacterium strain as described for use in the method. AT-DNA region to be introduced to a plant using this Agrobacterium strainfurther comprises a plasmid having a T-DNA region adjacent to at leastone Agrobacterium T-DNA border.

Although superbinary systems are known, for example, see WO 94/00977A1,WO 95/06722A1, and WO 95/16031A1, and are further described by Komari etal. (supra), and Komori et al. (supra), these systems possess a numberof disadvantages. An operational disadvantage of the superbinary system,which is overcome by the Agrobacterium strains and methods describedherein, is the necessity for formation of a co-integrant plasmid betweenpSB1 and pSB11 (and its derivatives) as the means by which the alteredT-DNA borne on pSB11 derivatives is to be stably maintained inAgrobacterium. This co-integration event generates a pair of large (ca.2.3 kbp) directly repeated sequences due to recombination between thehomologous regions of pSB1 and pSB11. As is well known to those skilledin the field of molecular biology, large repeated sequences such asthese are preferred targets for intramolecular recombination that leadseventually to DNA deletions and other rearrangements, particularly whenthe repeats are a part of plasmid structure. In the Agrobacteriumsuperbinary system, such rearrangements may lead to partialrearrangement or complete loss of the T-DNA region introduced by pSB11derivatives, ultimately resulting in little or no transfer of intactdesired foreign genes into the host plant cells.

A further disadvantage to the above-described superbinary system, andwhich is also overcome by the Agrobacterium strains and methodsdescribed herein, is that the formation of the co-integrant plasmidbetween pSB1 and pSB11 derivatives generates a large plasmid (minimally,greater than 43 kbp) having two distinct ColE1-type (incompatibilitygroup pMB1/ColE1) origins of replication (ori), as well as a third oriderived from the RK2 plasmid (incompatibility group IncP). Although innormal circumstances the ColE1 ori is nonfunctional in Agrobacterium,genomic mutations are known which allow the stable maintenance ofplasmids having a ColE1 ori in Agrobacterium (Ruslyakova et al. (1999)Russian J. Genet. 35:327-331). In cells having such mutations, a plasmidsuch as the pSB1::pSB11 derivative co-integrant having three functionalorigins of replication would be expected to be highly unstable. Thus,the superbinary system has imperfections that are advantageouslyaddressed by elements of the Agrobacterium strains and methods fortransforming plants described herein.

The DNA structure of the foreign gene or genes destined for introductionand expression in transgenic plant cells by Agrobacterium-mediatedtransformation can have a profound influence on the stability of thebinary vector plasmid or shuttle vector plasmid harboring those genes incells of Escherichia coli and Agrobacterium. Instability is particularlymanifested when the foreign genes comprise gene components that areemployed multiple times within the gene constructs. For example, it isnot uncommon that a particular plant-expressible promoter may be used todrive the expression of different protein coding regions in a transgenicplant. Other gene components such as 3′ untranslated regions (3′UTR)(i.e., transcription termination and polyadenylation additiondetermining sequences) and even highly similar protein coding regionsmay be duplicated or present in multiple copies within a single T-DNAregion. As mentioned above, these repeated sequence elements, which mayexist in either inverted or directly repeated orientations, are targetsfor intramolecular recombinations that may lead to DNA deletions andother rearrangements, particularly as the repeats are a part of plasmidstructure.

Multiple specialized strains of E. coli have been developed to serve asmolecular cloning hosts that help to overcome such instabilitydifficulties (e.g., STBL2™, STBL3™, and STBL4™ strains offered byINVITROGEN; Carlsbad, Calif.). A feature common to all such E. colicloning strains is the presence of a genomic mutation in a recA gene.The RecA protein is a multifunctional enzyme that plays a role inhomologous recombination, DNA repair, and induction of the bacterial SOSresponse. In the homologous recombination process, the protein functionsas a DNA-dependent ATPase, promoting synapsis, heteroduplex formationand strand exchange between homologous DNAs. Thus, cells deficient inRecA function are more prone to tolerate homologous DNA sequenceswithout rearrangement or deletion.

RecA-deficient strains of Agrobacterium have been developed to helpaddress the instability problems observed when cloning large DNAfragments containing repeated sequences (Klapwicj et al. (1979) Molec.Gen. Genet. 173:171-175; Farrand et al. (1989) J. Bacteriol.171:5314-5321; Lazo et al. (1991) Bio/Technology 9:963-967). Thesestrains have proven useful in helping stabilize high molecular weighttransforming constructs in some cases (Frary and Hamilton (2001),Transgenic Res. 10:121-132), but not in all instances (Song et al.(2003) Theor. Appl. Genet. 107:958-964). Thus, Agrobacterium chromosomalbackgrounds that are recA defective in developing strains that arehighly efficient in plasmid maintenance and plant transformationcapability can be advantageously used. In addition to usingAgrobacterium chromosomal backgrounds that are recA defective indeveloping strains for use in the methods described herein, the recAfunctionality can be deactivated in an existing or produced strain tomake that strain useful in the methods described herein. See, e.g.,Farrand et al. (supra). For example, a strain can be developed with RecAfunctionality and any chromosomal additions desired, e.g., the additionof vir genes, can be made then the RecA functionality disabled.

BIBAC vectors designed to enable efficient transformation of large DNAfragments into plant and non-plant host cells can be used. See, e.g.,U.S. Pat. No. 5,733,744, U.S. Pat. No. 5,977,439, and U.S. PatentApplication No. 2002/0123100A1. One Agrobacterium strain that can beutilized with the BIBAC vectors is the RecA-deficient strain UIA143developed by Farrand et al. (supra). Refinements to the BIBAC systemhave used subsets of the genes harbored on the 14.8 KpnI VirBCDGfragment in combination with other vir genes to enhance the planttransformation capability of engineered Agrobacterium strains. Inparticular, the virG gene from the 14.8 KpnI VirBCDG fragment has beenemployed alone or in combination with the virE1 and virE2 genes frompTiA6 in the UIA143 RecA-deficient strain. See, e.g., Hamilton et al.(1996) Proc. Natl. Acad. Sci. 93:9975-9979; Hamilton (1997), Gene200:107-116; Frary and Hamilton (supra).

In addition, a suitable vector used to transform plant cell using themethods described herein can contain a selectable marker gene encoding aprotein that confers on the transformed plant cells resistance to anantibiotic or a herbicide. The individually employed selectable markergene may accordingly permit the selection of transformed cells while thegrowth of cells that do not contain the inserted DNA can be suppressedby the selective compound. The particular selectable marker gene(s) usedmay depend on experimental design or preference, but any of thefollowing selectable markers may be used, as well as any other gene notlisted herein that could function as a selectable marker. Examples ofselectable markers include, but are not limited to, genes that provideresistance or tolerance to antibiotics such as Kanamycin, G418,Hygromycin, Bleomycin, and Methotrexate, or to herbicides, such asPhosphinothricin (Bialaphos), Glyphosate, Imidazolinones, Sulfonylureas,Triazolopyrimidines, Chlorosulfuron, Bromoxynil, and DALAPON.

In addition to a selectable marker, a reporter gene may also be used. Insome instances a reporter gene could be used without a selectablemarker. Reporter genes are genes that typically do not provide a growthadvantage to the recipient organism or tissue. Reporter genes typicallyencode for a protein that provides for a phenotypic change or enzymaticproperty. Suitable reporter genes include, but are not limited to, thosethat encode glucuronidase (GUS), firefly luciferase, or fluorescentproteins such as green fluorescent protein and yellow fluorescentprotein.

In addition to numerous technologies for transforming plants, the typeof tissue that is contacted with the foreign genes may vary as well.Such tissue may include, but is not limited to, embryogenic tissue,callus tissue types I and II, hypocotyl, and meristem. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques within the skill of the art. One skilled in the field ofplant transformation will understand that multiple methodologies areavailable for the production of transformed plants, and that they may bemodified and specialized to accommodate biological differences betweenvarious host plant species.

Regardless of the particular transformation technique employed, theforeign gene can be incorporated into a gene transfer vector adapted toexpress the foreign gene in a plant cell by including in the vector aplant promoter. In addition to plant promoters, promoters from a varietyof sources can be used efficiently in plant cells to express foreigngenes. For example, promoters of bacterial origin, such as the octopinesynthase promoter, the nopaline synthase promoter, the mannopinesynthase promoter; promoters of viral origin, such as the 35S and 19Spromoters of cauliflower mosaic virus (CaMV), a promoter from sugarcanebacilliform virus, and the like may be used. Plant-derived promotersinclude, but are not limited to, ribulose-1,6-bisphosphate (RUBP)carboxylase small subunit (ssu) promoter, beta-conglycinin promoter,phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat-shockpromoters, ADF (actin depolymerization factor) promoter, and tissuespecific promoters. Promoters may also contain certain enhancer sequenceelements that may improve the transcription efficiency. Typicalenhancers include, but are not limited to, alcohol dehydrogenase 1(ADH1) intron 1 and ADH1-intron 6. Constitutive promoters may be used.Constitutive promoters direct continuous gene expression in nearly allcells types and at nearly all times (e.g., actin promoter, ubiquitinpromoter, CaMV 35S promoter). Tissue specific promoters are responsiblefor gene expression in specific cell or tissue types, such as the leavesor seeds. Examples of other promoters that may be used include thosethat are active during a certain stage of the plant's development, aswell as active in specific plant tissues and organs. Examples of suchpromoters include, but are not limited to, promoters that are rootspecific, pollen-specific, embryo specific, corn silk specific, cottonfiber specific, seed endosperm specific, and phloem specific.

Under certain circumstances, the use of an inducible promoter may bedesirable. An inducible promoter is responsible for expression of genesin response to a specific signal, such as physical stimulus (e.g., heatshock gene promoters); light (e.g., Ribulose-bis-phosphate 1,5carboxylase promoter); hormone (e.g., glucocorticoid); antibiotic (e.g.,Tetracycline); metabolites; and stress (e.g., drought). Other desirabletranscription and translation elements that function in plants also maybe used, such as, for example, 5′ untranslated leader sequences, RNAtranscription termination sequences and poly-adenylate addition signalsequences. Any suitable plant-specific gene transfer vector known to theart may be used.

Transgenic crops containing insect resistance (IR) traits are prevalentin corn and cotton plants throughout North America, and usage of thesetraits is expanding globally. Commercial transgenic crops combining IRand herbicide tolerance (HT) traits have been developed by multiple seedcompanies. These include combinations of IR traits conferred by Bt(Bacillus thuringiensis) insecticidal proteins and HT traits such astolerance to Acetolactate Synthase (ALS) inhibitors such asSulfonylureas, Imidazolinones, Triazolopyrimidine, Sulfonanilides, andthe like, Glutamine Synthetase (GS) inhibitors such as Bialaphos,Glufosinate, and the like, 4-HydroxyPhenylPyruvate Dioxygenase (HPPD)inhibitors such as Mesotrione, Isoxaflutole, and the like,5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such asGlyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase)inhibitors such as Haloxyfop, Quizalofop, Diclofop, and the like. Otherexamples are known in which transgenically provided proteins provideplant tolerance to herbicide chemical classes such as phenoxy acidsherbicides and pyridyloxyacetates auxin herbicides (see WO2007/053482A2), or phenoxy acids herbicides andaryloxyphenoxypropionates herbicides (see WO 2005107437A2,A3). Theability to control multiple pest problems through IR traits is avaluable commercial product concept, and the convenience of this productconcept is enhanced if insect control traits and weed control traits arecombined in the same plant. Further, improved value may be obtained viasingle plant combinations of IR traits conferred by a Bt insecticidalprotein with one or more additional HT traits such as those mentionedabove, plus one or more additional input traits (e.g., other insectresistance conferred by Bt-derived or other insecticidal proteins,insect resistance conferred by mechanisms such as RNAi and the like,disease resistance, stress tolerance, improved nitrogen utilization, andthe like), or output traits (e.g., high oils content, healthy oilcomposition, nutritional improvement, and the like). Such combinationsmay be obtained either through conventional breeding (e.g., breedingstack) or jointly as a novel transformation event involving thesimultaneous introduction of multiple genes (e.g., molecular stack).Benefits include the ability to manage insect pests and improved weedcontrol in a crop plant that provides secondary benefits to the producerand/or the consumer. Thus, the Agrobacterium strains and methodsdescribed herein can be used to provide transformed plants withcombinations of traits that comprise a complete agronomic package ofimproved crop quality with the ability to flexibly and cost effectivelycontrol any number of agronomic issues.

The virG genes of various pTi plasmids have been studied to understandtheir ability to enhance plant transformation frequency. Liu et al.(1992, Plant Molec. Biol. 20:1071-1087) found that extra copies of virGgenes from multiple sources (i.e., from different pTi plasmids, butincluding pTiBo542) enhanced the transient transformation of someplants, and the magnitude of the effect depended on the identity of thehelper pTi plasmid with which the particular virG gene was paired. Amutant of a virG gene (presumably from pTiA6), named virGN54D (themutation replaces amino acid Asn54 with Asp), is constitutivelyexpressed in Agrobacterium (induction of wild-type virG genes requiresan acidic pH, a high monosaccharide concentration, and the presence ofphenolic inducers, such as acetosyringone). See Pazour et al. (1992) J.Bacteriol. 174:4169-4174. VirGN54D of pTiA6 was effective in enhancingmaize transformation, whereas multiple copies of the parent wild-typevirG were ineffective. See Hansen et al. (1994) J. Bacteriol.174:4169-4174. A “ternary” (i.e., three-plasmid) system wherein a copyof the constitutive mutant virGN54D gene from pTi15955 was co-residenton a pBBR1-derived plasmid in Agrobacterium tumefaciens strain LBA4404that contained the disarmed pTi helper plasmid pAL4404 and a binaryvector harboring genes for plant transformation has been described. Seevan der Fits et al. (2000) Plant Molec. Biol. 43:495-502. Theconstitutively expressed virGN54D gene was found to dramaticallyincrease both transient and stable transformation efficiencies ofseveral plant species. Plasmids containing the pBRR1 replication controlregion cannot be classed as belonging to any known incompatibility groupand, thus, may co-exist with a broad range of other plasmids in a singlehost. Further, the abilities of various combinations of vir genes toaffect plant transformation efficiencies in tobacco, cotton and ricehave been tested, specifically: the mutant virGN54D gene derived frompTiA6, the virG gene from pTiBo542, the VirE1/E2 genes from pTiA6, and acombination of the latter two gene sets. See Park et al. (2000) Theor.Appl. Genet. 101:1015-1020. Increases in transformation efficiencieswere observed with some plant species and additional copies of virgenes.

European Patent Application No. 2042602A1 and U.S. Patent ApplicationNo. 2010/0132068A1 describe cosmid binary vectors and “booster” plasmidsthat, when present in an Agrobacterium cell harboring a pTi helperplasmid, constitute further examples of ternary plasmid systems. Boosterplasmids as disclosed therein possess a replication origin of the IncWincompatibility group, and comprise plasmid pVGW, having the virGN54Dgene, and plasmid pVGW2, which is a derivative of pVGW havingmodifications to facilitate cloning and selection.

The functions encoded by chromosomal genes in Agrobacterium haveclassically been determined by two genetic approaches. The first, orforward genetics method, entails obtaining a molecular clone of the geneto be studied, followed by placement of the cloned gene in a geneticenvironment wherein a “gain of function” phenotype can be assessed. Asecond, or “reverse genetics method,” requires disruption of the genes'structure by insertion or deletion of sequences in or around the gene inthe chromosome, followed by determination of which proteins orphenotypes have been removed by the loss of gene function. This is theapproach used to construct the previously described RecA-deficientmutant of strain C58. See Farrand et al., supra. Those skilled in thefield of genetic manipulation of Agrobacterium cells will understandthat diverse vectors and numerous methods have been described to enablesuch gene disruption experiments. The method has proven to beparticularly useful when used to identify genes that are not involved invitality, growth, and plant transformation capability of the mutatedstrain. One such genetic locus in Agrobacterium strain C58 is thepgl/picA locus. See, Lee et al. (2001) Plant Microbe Interact.14:577-579; and Lee (2006) in Methods in Molecular Biology (K. Wang,ed.) No. 343: Agrobacterium Protocols (2nd Edition, Vol. 1) HUMANA PRESSInc., Totowa, N.J. pp. 55-66. Cells in which a virD2 gene has beenintegrated into this chromosomal locus by homologous recombination werefound to have a plant transformation phenotype identical to thatresulting from A. tumefaciens strains harboring the virD2 gene locatedon a replicating plasmid. See Lee et al., supra. Further, a T-DNA regionintegrated into the pgl/picA locus of C58 may be functionally deliveredto the plant cell (Oltmanns et al. (2010) Plant Physiol. 152:1158-1166).Thus, in strain C58, the pgl/picA locus can serve as a “neutralintegration site” for introduction of genes into the C58 chromosome. Asused herein, “neutral integration site” refers to a gene or chromosomallocus, natively present on the chromosome of an Agrobacterium cell,whose normal function is not required for the growth of the cell or forthe capability of the cell to perform all the functions required forplant transformation. When disrupted by the integration of a DNAsequence not normally present within that gene, the cell harboring adisrupted neutral integration site gene can productively perform planttransformation. By way of example, Hoekema et al. (1984, EMBO J.3:2485-2490) demonstrated that a functional T-region integrated into anuncharacterized locus in the C58 chromosome by means of Tn3transposition was productively transferred to plant cells.

The Agrobacterium strains discussed herein can be used advantageously tointroduce one or more genes into a plant, e.g., to provide individual ormultiple insecticidal or herbicidal properties to the plant. Forexample, the Agrobacterium strains can be used to introduce one or more,two or more, three or more, four or more, five or more, or six or moregenes into a plant. Using the Agrobacterium strains described herein,the polynucleotide containing the selectable gene sequences is insertedinto a single location in the plant cell when the plant cell istransformed. In terms of the size of the T-DNA regions used to insertthe genes, the T-DNA regions can be equal to or greater than 15,000nucleotide base pairs, greater than or equal to 20,000 nucleotide basepairs, equal to or greater than 25,000 nucleotide base pairs, equal toor greater than 26,000 nucleotide base pairs, equal to or greater than27,000 nucleotide base pairs, equal to or greater than 28,000 nucleotidebase pairs, equal to or greater than 29,000 nucleotide base pairs, orequal to or greater than 30,000 nucleotide base pairs. When using theAgrobacterium strains described herein, the selectable gene sequencescan have equal to or greater than 60%, equal to or greater than 65%,equal to or greater than 67%, equal to or greater than 69.5%, equal toor greater than 70%, equal to or greater than 75%, or equal to orgreater than 80% sequence homology and retain their transcribablesequence identities. The types of genes that can be introduced canencode insecticidal proteins, herbicidal proteins, or a mixture ofinsecticidal proteins and herbicidal proteins. Specific examples ofgenes that can be introduced include the genes encoding the Cry1Cainsecticidal protein, Cry1F insecticidal protein, Cry1Ab1 insecticidalprotein, and AAD1 herbicidal protein, which can be introduced in variouscombinations or as a set including all four. Monocotyledonous (monocot)and dicotyledonous (dicot) species can be transformed using theseAgrobacterium strains.

Also disclosed herein is the nilA genomic locus of Agrobacteriumtumefaciens, into which a polynucleotide sequence can be integrated.Such an integrated polynucleotide sequence can include any vir gene orvir operon or other useful genes. Examples 17-20 show theidentification, characterization, and use of the nilA genomic locus ofAgrobacterium tumefaciens as well as the production of an Agrobacteriumtumefaciens strain with multiple vir genes located on the chromosome.The nilA genomic locus, or any locus which shares 85-100% nucleotidesequence identity, could be identified in other Agrobacterium strainsusing the techniques for identification and characterization describedherein, and any such identified nilA loci could be used in a mannersimilar to that described herein to integrate vir or other suitablegenes which can, e.g., increase the efficiency of plant transformation.The techniques for identification and characterization of such a genomiclocus described herein could also be used to identify other neutralintegration sites on the Agrobacterium chromosome at whichpolynucleotide sequences containing vir or other genes can be integratedsuch that the Agrobacterium strain remains capable of transformingplants. Some chromosomal sites are already known that could be used asneutral integration sites, for example, the RecA site in aRecA-deficient strain, and the pgl/picA locus in Agrobacteriumtumefaciens strain C58. However, there is a need to identify new neutralsites in Agrobacterium tumefaciens strains besides C58, as the pgl/picAlocus is not detected in some other strains, for example, strain LBA4404(Oltmanns et al., supra). Additional chromosomal sites which can be usedas neutral integration sites are described in U.S. Pat. No. 6,323,396.Thus, an Agrobacterium strain with a vir gene integrated into a neutralintegration site on the Agrobacterium chromosome is also disclosed. Suchan Agrobacterium strain could use a nilA genomic locus or other neutralintegration site for the integration of vir genes.

Multiple types of useful genes could be added to the chromosome in thisway making the use of T-helper plasmids unnecessary. For example,additional vir genes and multiple copies of useful vir genes fromdifferent strains could be used.

Also disclosed herein is an Agrobacterium strain containing vir genes ona helper plasmid having a replication origin of an incompatibility groupother than IncP and a plasmid having a T-DNA region adjacent to at leastone Agrobacterium T-DNA border, the plasmid having a replication originof an IncP incompatibility group.

Further disclosed are plants made by the methods described herein usingthe Agrobacterium strains described herein. Such plants stably integrateany T-DNA regions introduced using the methods described herein.Further, such plants express any genes and exhibit any genetic traitsconferred by those T-DNA regions. Additionally, any progeny of theplants made by the methods described herein using the Agrobacteriumstrains described herein stably produce any genes and exhibit anygenetic traits conferred by those T-DNA regions found in the parent.

In a specific embodiment, a plant is described that stably expressesCry1Ca insecticidal proteins, Cry1F insecticidal proteins, Cry1Ab1insecticidal proteins, and AAD1 herbicidal proteins. This plant, forexample, can be maize.

While certain example Agrobacterium strains are described herein, thefunctionality discussed could be moved to other Agrobacterium strainswith the same criteria, e.g., other strains which are deficient in RecAor could be made deficient in RecA. Examples of other strains that couldbe used with the strains and methods described herein include, but arenot limited to, Agrobacterium tumefaciens strain C58, Agrobacteriumtumefaciens strain Chry5, Agrobacterium rhizogenes strains,Agrobacterium tumefaciens strain EHA101, Agrobacterium tumefaciensstrain EHA105, Agrobacterium tumefaciens strain MOG101, andAgrobacterium tumefaciens strain T37.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for utilizing theAgrobacterium strains and practicing the methods described herein. Theseexamples should not be construed as limiting. All percentages are byweight and all solvent mixture proportions are by volume unlessotherwise noted. All temperatures are in degrees Celsius.

Unless specifically indicated or implied, the terms “a,” “an,” and “the”signify “at least one” as used herein.

Example 1: Construction of a Deletion Variant of Plasmid pUCD2

Construction of plasmid pUCD2 was described by Close et al. (1984,Plasmid 12:111-118), and the complete 13,239 bp DNA sequence isdisclosed for the first time herein as SEQ ID NO:1. pUCD2 harbors fourgenes conferring bacterial resistance to antibiotics: specifically,resistance to Spectinomycin, Kanamycin, Tetracycline, and Ampicillin(FIG. 1). Standard molecular biology methods, as taught, for example, inSambrook et al. (1989, Molecular Cloning: A Laboratory Manual (2ndEdition, COLD SPRING HARBOR LABORATORY PRESS, Plainview, N.Y.) andAusubel et al. (1995, Current Protocols in Molecular Biology (GREENEPUBLISHING AND WILEY-INTERSCIENCE, New York), and updates thereof, wereemployed in this and other steps described in this example and in otherexamples of this disclosure. A first modification to pUCD2 was made bycleaving pUCD2 DNA with restriction enzymes Sac I and Sac II andligation to a mostly double-stranded oligonucleotide fragment havingappropriate overhanging “sticky ends” compatible with Sac I- or Sac IIgenerated overhangs. This double-stranded oligonucleotide (FIG. 1) wascreated by annealing two complementary oligonucleotide sequences,disclosed as SEQ ID NO:2 and SEQ ID NO:3. The sequences of theoligonucleotides of SEQ ID NO:2 and SEQ ID NO:3 are designed to restorea functional Kanamycin resistance gene upon ligation with pUCD2 DNAcleaved with Sac I and Sac II. This manipulation created plasmidpDAB9290 (FIG. 1), which differs from pUCD2 by the deletion of thecoding region for Spectinomycin resistance, elimination of a Kpn Irestriction enzyme recognition site from within the coding region forKanamycin resistance, and creation of a new Kpn I site downstream of theKanamycin resistance coding region.

DNA of plasmid pDAB9290 was further manipulated to render inoperativethe genes encoding Tetracycline resistance and Ampicillin resistance byfirst cleaving with restriction enzymes Pst I and Sal I, treating theoverhanging ends left by these enzymes with the QUICK BLUNTING™ kit (NEWENGLAND BIOLABS; Ipswich, Mass.) to create blunt ends, and self ligationto circularize the fragments thus produced. The resulting plasmid(pDAB9291) retains only the Kanamycin bacterial antibiotic resistancegene, and has a unique site for cleavage by Kpn I downstream of theKanamycin resistance gene. The sequence of pDAB9291 is disclosed as SEQID NO:4. Plasmid pDAB9291 has two origins of replication, one (colE1incompatibility group) derived from plasmid pBR322, and a second derivedfrom plasmid pSa (incompatibility group W). Thus, plasmid pDAB9291 iscapable of medium-copy-number maintenance in E. coli and Agrobacterium.

Example 2: Cloning of a 14.8 Kpn I virBCDG Fragment into pDAB9291

A 14.8-kbp Kpn I fragment containing the virG, virB, and virC operonsand virD1 from the “supervirulent” pTiBo542 (FIG. 1) was isolated fromplasmid pSB1 (Komari et al., supra; and Komori et al., supra), andcloned into the unique Kpn I site of pDAB9291. Plasmids containing eachof the two possible orientations of the insert fragment were obtained,and were named pDAB9292 and pDAB9293. One plasmid, pDAB9292 (FIG. 1) wasselected for further work. The DNA sequence of pDAB9292 is disclosed asSEQ ID NO:5.

Example 3: Construction of a RecA-Deficient Agrobacterium StrainHarboring the Helper Plasmid pTiEHA105

Agrobacterium strain UIA143 is a RecA-deficient strain having the C58genetic background and was constructed and described by Farrand et al.(supra). The chromosomal recA gene was deleted and replaced with a genecassette conferring resistance to Erythromycin at 150 μg/mL. The UIA143strain contains no Ti plasmid or Ti plasmid derivative.

Agrobacterium strain EHA105, constructed and described by Hood et al.(1993, Transgenic Research 2:208-221), harbors a helper plasmid (hereincalled pTiEHA105) derived from the “supervirulent” pTiBo542 plasmid.Plasmid pTiEHA105 DNA was prepared from strain EHA105 and introduced byelectroporation into cells of strain UIA143 made electrocompetent bystandard methods (Weigel and Glazebrook (2002) Arabidopsis: A LaboratoryManual, COLD SPRING HARBOR PRESS, Cold Spring Harbor, N.Y., 354 pages;Mersereau et al. (1990) Gene 90:149-151; Mattanovich, et al. (1989)Nucl. Acids Res. 17:6747)). Strain UIA143 cells transformed withpTiEHA105 were selected by their ability to grow on AB minimal medium(Watson, et al. (1975) J. Bacteriol. 123:255-264) using purified agarand mannopine (2 mg/mL) as a sole source of carbon and nitrogen forgrowth (Guyon et al. (1980) Proc. Natl. Acad. Sci. 77:2693-2697; Dessauxet al. (1987) Molec. Gen. Genet. 208:301-308).

The presence of pTiEHA105 was verified by polymerase chain reaction(PCR) using primers designed to amplify fragments of the pTiBo542 virD2and virG genes, and further characterized by Southern blot analysis oftotal DNA prepared from candidate colonies probed with 32P-labeled DNAof pTiEHA101 purified by cesium chloride gradient centrifugation. ThisAgrobacterium strain (i.e., UIA143 containing pTiEHA105) is namedDA2552.

Example 4: Construction of a RecA-Deficient Agrobacterium StrainHarboring the Helper Plasmid pTiC58Δ

Strain Z707 was derived by replacing the entire T-DNA region of thepTiC58 plasmid of Agrobacterium tumefaciens strain C58 with the npt Igene of Tn903, which confers resistance to Kanamycin. The entire virregion of the resulting plasmid, herein called pTiC58Δ, was left intact(Hepburn et al. (1985) J. Gen. Microbiol. 131:2961-2969). The helperplasmid pTiC58Δ from strain Z707 was purified by cesium chloridegradient centrifugation and was electroporated into electrocompetentUIA143 cells. A transformant was selected on the basis of the pTiC58Δplasmid-borne Kanamycin resistance gene and chromosomally borneErythromycin resistance gene, and the strain was named DA2569. Presenceof pTiC58Δ in DA2569 was verified by PCR amplification using primers todetect selected vir gene regions and by Southern blot analysis of totalDNA prepared from DA2569 candidate colonies probed with 32P-labeled DNAof pTiC58Δ purified by cesium chloride gradient centrifugation fromcells of strain Z707.

Example 5: Construction of a RecA-Deficient Agrobacterium StrainHarboring the Helper Plasmid pMP90

Agrobacterium tumefaciens strain GV3101(pMP90) harbors a deleted versionof pTiC58 called pMP90, from which the entire T-DNA region has beendeleted and replaced with a gene conferring resistance to Gentamicin(Koncz and Schell (1986) Mol. Gen. Genet. 204:383-396). DNA of plasmidpMP90 is prepared by methods such as cesium chloride gradientcentrifugation or the MACHEREY-NAGEL NUCLEOBOND XTRA MAXI KIT “LOW COPY”(MACHEREY-NAGEL Inc.; Bethelem, Pa.) and is electroporated into UIA143cells. A transformant is selected on the basis of the pMP90plasmid-borne Gentamicin resistance gene (100 μg/mL) and the strain isnamed DAt20538. Presence of pMP90 in DAt20538 is verified by PCRamplification using primers to detect selected vir gene regions and bySouthern blot analysis of total DNA prepared from DAt20538.

Example 6: Construction of a RecA-Deficient Agrobacterium StrainHarboring the Helper Plasmid pMP90RK

The helper plasmid pMP90 described in Example 5 was further modified bythe introduction (via double crossover homologous recombination) of a42-kbp EcoR I fragment derived from plasmid pRK2013 (Figurski andHelinski (1979) Proc. Natl. Acad. Sci. USA 79:1648-1652). The 42-kbpfragment contains plasmid RK2-derived genes for plasmid replication andmobilization (e.g., trfA, tra1, tra2, tra3, and oriT), and a geneconferring resistance to Kanamycin. This manipulation replaced theGentamicin resistance gene of plasmid pMP90, and the resulting plasmidwas named pMP90RK (Koncz and Schell, supra). DNA of plasmid pMP90RK isprepared by methods such as cesium chloride gradient centrifugation orthe MACHEREY-NAGEL NUCLEOBOND XTRA MAXI KIT “LOW COPY” and iselectroporated into electrocompetent UTA143 cells. A transformant isselected on the basis of the pMP90RK plasmid-borne Kanamycin resistancegene and the strain is named DAt20539. Presence of pMP90RK in DAt20539is verified by PCR amplification using primers to detect selected virgene regions and by Southern blot analysis of total DNA prepared fromDAt20539.

Example 7: Electroporation of pDAB9292 DNA into Agrobacterium StrainDA2552

Electrocompetent DA2552 cells were prepared using a standard protocol(see Example 3). 50 μL of the competent DA2552 cells were thawed on iceand were transformed using 300 to 400 ng of plasmid pDAB9292 DNA. TheDNA and cell mix was electroporated using prechilled electroporationcuvettes (0.2 cm) and a BIO-RAD GENE PULSER electroporator (BIO-RADInc.; Hercules, Calif.) with the following conditions: Voltage: 2.5 kV,Pulse length: 5 msec, Capacitance output: 25 μFarad, Resistance: 200ohms. After electroporation, 1 mL of YEP (gm/L: Yeast Extract 10,Peptone 10, NaCl 5) broth was added to the cuvette and the cell-YEPsuspension was transferred to a 15 mL culture tube. The cells wereincubated at 28° C. with gentle agitation for four hours after which theculture was plated on YEP+ agar containing Kanamycin at 50 μg/mL andErythromycin at 150 μg/mL. The plates were incubated for two to fourdays at 28° C. and colonies were selected and streaked onto fresh YEP+agar plates with antibiotics as above and incubated at 28° C. for one tothree days. These colonies were verified as Agrobacterium using theketolactose test (Bouzar et al. (1995) in Methods in Molecular Biology(K. Gartland and M. Davey, eds.) Agrobacterium Protocols (Vol. 44)HUMANA PRESS, Totowa, N.J. pp. 9-13. Several ketolactose positivecolonies were selected to start 3 mL YEP (with antibiotics) seedcultures that were grown overnight at 28° C. while shaking. 300 μL ofeach seed culture was used to inoculate a 200 mL YEP (with antibiotics)overnight culture grown at 28° C. while shaking at 200 rpm. Plasmid DNAwas prepared from 165 mL of each 200 mL overnight culture using aMACHEREY-NAGEL NUCLEOBOND® XTRA MAXI PLASMID DNA PURIFICATION kit. Themanufacturer's protocol was followed, except 30 mL each of buffer RES,LYS, and NEU was used. The eluted DNA was stored at 4° C.

Restriction enzyme digestion of the plasmid DNA with BamH I was used tovalidate the presence of pDAB9292 in these isolates, and colonies havingthe correct patterns were then further purified using two passages ofsingle colony isolation. Plasmid DNA was prepared from overnightcultures as described above and restriction digest analysis was used toverify the presence of the intact pDAB9292. Plasmid DNA of the pDAB9292vector originally used in the DA2552 transformation was included as adigested standard. Four separate digest reactions (Pst I, BamH I, Mfe Iand Hind III) were run using 750 ng to 1 μg of DNA. The reaction wasallowed to run one to two hours and was analyzed by agarose gelelectrophoresis (0.8% w/v) and the DNA fragments were visualized byethidium bromide staining. This Agrobacterium strain (i.e., DA2552harboring pDAB9292) is named DAt13192. This strain provides the basisfor a recombination-deficient “ternary” plant transformation system.

Example 8: Electroporation of pDAB9292 DNA into Agrobacterium StrainGV3101(pMP90)

Cells of Agrobacterium tumefaciens strain GV3101(pMP90) (Koncz andSchell, supra) were made electrocompetent by a standard protocol (seeExample 3). 50 μL of the competent GV3101(pMP90) cells were thawed onice and were transformed using 300 to 400 ng of plasmid pDAB9292 DNA.The DNA and cell mix was electroporated using prechilled electroporationcuvettes (0.2 cm) and a BIO-RAD GENE PULSER electroporator with thefollowing conditions: Voltage: 2.5 kV, Pulse length: 5 msec, Capacitanceoutput: 25 μFarad, Resistance: 200 ohms. After electroporation, 1 mL ofYEP broth was added to the cuvette and the cell-YEP suspension wastransferred to a 15 mL culture tube. The cells were incubated at 28° C.with gentle agitation for four hours after which the culture was platedon YEP+ agar containing Kanamycin at 50 μg/mL and Gentamicin at 100μg/mL. The plates were incubated for two to four days at 28° C. andcolonies were selected and streaked onto fresh YEP+ agar plates withantibiotics as above and incubated at 28° C. for one to three days.These colonies were verified as Agrobacterium using the ketolactosetest. Several ketolactose positive colonies were selected to start 3 mLYEP (with antibiotics) seed cultures that were grown overnight at 28° C.while shaking. 300 μL of each seed culture was used to inoculate a 200mL YEP (with antibiotics) overnight culture grown at 28° C. whileshaking at 200 rpm. Plasmid DNA was prepared from 165 mL of each 200 mLovernight culture using a MACHEREY-NAGEL NUCLEOBOND® XTRA MAXI PLASMIDDNA PURIFICATION. The manufacturer's protocol was followed, except 30 mLeach of buffer RES, LYS and NEU was used. The eluted DNA was stored at4° C.

Restriction enzyme digestion of the plasmid DNA with BamH I was used tovalidate the presence of pDAB9292 in these isolates, and colonies havingthe correct patterns were then further purified using two passages ofsingle colony isolation. Plasmid DNA was prepared from overnightcultures as described above and restriction digest analysis was used toverify the presence of the intact pDAB9292. Plasmid DNA of the pDAB9292vector originally used in the GV3101(pMP90) transformation was includedas a digested standard. Four separate digest reactions (Pst I, BamHI,Mfe I and Hind III) were run using 750 ng to 1 μg of DNA. The reactionwas allowed to run one to two hours and was analyzed by agarose gelelectrophoresis (0.8% w/v) and the DNA fragments were visualized byethidium bromide staining. The A. tumefaciens GV3101 isolate harboringthe pMP90 Ti helper plasmid and pDAB9292 is called DAt20712.

Example 9: Electroporation of pDAB9292 DNA into Agrobacterium StrainLBA4404

Cells of Agrobacterium tumefaciens strain LBA4404 (Ooms et al. (1982)Plasmid 7:15-29) were made electrocompetent by a standard protocol (seeExample 3). 50 μL of the competent LBA4404 cells were thawed on ice andwere transformed using 300 to 400 ng of plasmid pDAB9292 DNA. The DNAand cell mix was electroporated using prechilled electroporationcuvettes (0.2 cm) and a BIO-RAD GENE PULSER electroporator with thefollowing conditions: Voltage: 2.5 kV, Pulse length: 5 msec, Capacitanceoutput: 25 μFarad, Resistance: 200 ohms. After electroporation, 1 mL ofYEP broth was added to the cuvette and the cell-YEP suspension wastransferred to a 15 mL culture tube. The cells were incubated at 28° C.with gentle agitation for four hours after which the culture was platedon YEP+ agar containing Kanamycin at 50 μg/mL and Streptomycin at 250μg/mL. The plates were incubated for two to four days at 28° C. andcolonies were selected and streaked onto fresh YEP+ agar plates withantibiotics as above and incubated at 28° C. for one to three days.These colonies were verified as Agrobacterium using the ketolactose testand were further purified using two passages of single colony isolation.

Several ketolactose positive colonies were selected to start 3 mL YEP(with antibiotics) seed cultures that were grown overnight at 28° C.while shaking. 300 μL of each seed culture was used to inoculate a 200mL YEP (with antibiotics) overnight culture grown at 28° C. whileshaking at 200 rpm. Plasmid DNA was prepared from 165 mL of each 200 mLovernight culture using a MACHEREY-NAGEL NUCLEOBOND® XTRA MAXI PLASMIDDNA PURIFICATION kit. The manufacturer's protocol was followed, except30 mL each of buffer RES, LYS and NEU was used. The eluted DNA wasstored at 4° C.

The presence of the intact pDAB9292 plasmid was verified by restrictiondigest analysis. Plasmid DNA of the pDAB9292 vector originally used inthe LBA4404 transformation was included as a digested standard. Threeseparate digest reactions (Pst I, BamH I, and Hind III) were run using750 ng to 1 μg of DNA. The reaction was allowed to run one to two hoursand was analyzed by agarose gel electrophoresis (0.8% w/v) and the DNAfragments were visualized by ethidium bromide staining. The A.tumefaciens LBA4404 isolate harboring pDAB9292 is called DAt20711. Thisstrain provides the basis for a recombination-proficient “ternary”system.

Example 10: Electroporation of pDAB9292 DNA into Agrobacterium StrainDAt20538

Electrocompetent DAt20538 cells are prepared using a standard protocol(see Example 3). 50 μL of competent DAt20538 cells are thawed on ice andare transformed using 300 to 400 ng of plasmid pDAB9292 DNA. The DNA andcell mix is electroporated using prechilled electroporation cuvettes(0.2 cm) and a BIO-RAD GENE PULSER electroporator with the followingconditions: Voltage: 2.5 kV, Pulse length: 5 msec, Capacitance output:25 μFarad, Resistance: 200 ohms. After electroporation, 1 mL of YEPbroth are added to the cuvette and the cell-YEP suspension istransferred to a 15 mL culture tube. The cells are incubated at 28° C.with gentle agitation for four hours after which the culture is platedon YEP+ agar containing Kanamycin at 50 μg/mL and Gentamicin at 100μg/mL. The plates are incubated for two to four days at 28° C. andcolonies are selected and streaked onto fresh YEP+ agar plates withantibiotics as above and incubated at 28° C. for one to three days.These colonies are verified as Agrobacterium using the ketolactose testand ketolactose positive colonies are further isolated using twopassages of single colony isolation.

Colonies are selected to start 3 mL YEP (with antibiotics) seed culturesthat are grown overnight at 28° C. while shaking. 300 μL of each seedculture is used to inoculate a 200 mL YEP (with antibiotics) overnightculture grown at 28° C. while shaking at 200 rpm. Plasmid DNA isprepared from 165 mL of each 200 mL overnight culture using aMACHEREY-NAGEL NUCLEOBOND® XTRA MAXI PLASMID DNA PURIFICATION kit. Themanufacturer's protocol is followed, except 30 mL each of buffer RES,LYS and NEU are used. The eluted DNA is stored at 4° C.

Restriction digest analysis is used to verify the presence of the intactpDAB9292 plasmid. Plasmid DNA of the pDAB9292 vector originally used inthe DAt20538 transformation is included as a digested standard. Fourseparate digest reactions such as Pst I, BamHI, Mfe I and Hind III arerun using 750 ng to 1 μg of DNA. The reaction is allowed to run one totwo hours and is analyzed by agarose gel electrophoresis (0.8% w/v) andthe DNA fragments are visualized by ethidium bromide staining. The A.tumefaciens DAt20538 isolate harboring pDAB9292 is calledDAt20538(pDAB9292).

Example 11: Construction of Plant Transformation Vectors Having MultipleRepeated Sequence Elements and Introduction into Agrobacterium Strains

The utility of an engineered Agrobacterium tumefaciens strain having adeficiency in RecA function in combination with the auxiliary vir genesprovided by the 14.8 KpnI VirBCDG fragment is illustrated herein. Abinary plant transformation vector, pDAB101513 (FIG. 4A), wasconstructed in E. coli cloning strain STBL2™ by a combination ofstandard cloning methods (as described, for example, in Sambrook et al.(1989, supra) and Ausubel et al. (1995, supra)) and GATEWAY™ technology(INVITROGEN). Binary vector pDAB101513 is based on the IncP-typereplication origin of plasmid RK2, and the vector backbone harbors abacterial gene conferring resistance to Spectinomycin (SpcR in FIG. 4)at 100 μg/mL. The T-DNA border repeats are derived from the TL region ofpTi15955. Within the Right Border (T-DNA Border B in FIG. 4) and tripleLeft Borders (T-DNA Border A in FIG. 4) of the T-DNA region of plasmidpDAB101513 are positioned four plant-expressible, plant-codon-optimizedprotein coding sequences (CDS), the transcription of each one beingdriven by a 1,991 bp maize ubiquitin1 promoter with associated intron1(U.S. Pat. No. 5,510,474). Three of the coding regions encode separateBt Cry1 proteins (Cry1Ca, SEQ ID NO:7; Cry1Fa, SEQ ID NO:9; and Cry1Ab,SEQ ID NO:11), each comprising around 3,500 bp. These coding regionswere codon optimized for expression in maize plants using a maize (Zeamays) codon bias table calculated from analysis of 706 maize proteincoding regions obtained from GENBANK deposits. Additional guidanceregarding the design and production of synthetic genes can be found in,for example, WO 97/13402A1, U.S. Pat. No. 6,166,302, and U.S. Pat. No.5,380,831. The three B.t protein coding regions are related to oneanother in the following fashion: The coding region for cry1Ca (SEQ IDNO:6) and the coding region for cry1Fa (SEQ ID NO:8) share 67% sequencehomology; the coding regions for cry1Ca (SEQ ID NO:6) and cry1Ab (SEQ IDNO:10) share 69.5% sequence homology, and the coding regions for cry1Fa(SEQ ID NO:8) and cry1Ab (SEQ ID NO:10) share 67% sequence homology.Further, the C-terminal 1,600 bp of the CDS for cry1Ca, cry1Fa, andcry1Ab share 73% sequence homology. Each of these three coding regionsis terminated by a 365 bp maize Per5 3′ Untranslated Region (3′UTR)(U.S. Pat. No. 6,384,207). The fourth gene comprises aplant-codon-optimized aad1 coding region (SEQ ID NO:12) that encodes theAAD1 selectable marker protein (SEQ ID NO:13) (U.S. Pat. No. 7,838,733)The aad1 coding region is not related to the CDS for cry1Ca, cry1Fa, orcry1Ab. The coding region for aad1 was designed using a plant-codon biastable. A maize codon bias table was calculated from 706 maize proteincoding sequences obtained from sequences deposited in GENBANK. Codonusage tables for tobacco (Nicotiana tabacum, 1268 CDS), canola (Brassicanapus, 530 CDS), cotton (Gossypium hirsutum, 197 CDS), and soybean(Glycine max; ca. 1000 CDS) were downloaded from data at the websitehttp://www.kazusa.or.jp/codon/. A biased codon set that comprisesfrequently used codons common to both maize and dicot datasets, inappropriate rescaled average relative amounts, was calculated afteromitting any redundant codon used less than about 10% of total codonuses for that amino acid in either plant type. The aad1 gene isterminated by a maize Lipase 3′UTR (U.S. Pat. No. 7,179,902). Thus,within the 22,729 bp T-DNA region of pDAB101513, the four copies of themaize ubi1 promoter comprise a total of 7,964 bases arranged in fourdirect repeats of almost 2 kbp (kilobase pairs) each, with each repeatbeing 100% related to the other. The three copies of the Per5 3′UTRcomprise a total of 1,095 bases arranged in three direct repeat units,each one being 100% related to the other, and the three coding regionscry1Ca, cry1Fa, and cry1Ab are arranged as direct repeats having between67% and 73% homology to one another. In total, the T-region ofpDAB101513 comprises about 86% highly repeated sequences, and may beconveniently illustrated below:

-   -   RB>Ubi1 promoter:cry1Ca CDS:Per5 3′UTR>Ubi1 promoter:cry1Fa        CDS:Per5 3′UTR>Ubi1 promoter:cry1Ab CDS:Per5 3′UTR>Ubi1        promoter:aad1 CDS:Lip 3′UTR>LB

The highly repeated nature of this construct required that the cloningsteps be completed in the E. coli cloning strain STBL2™, which isspecially engineered to maintain the integrity of clones containing suchhighly repeated DNA sequences.

Plasmid pDAB101513 was introduced by electroporation intoelectrocompetent cells ofA. tumefaciens strain EHA105 (renderedStreptomycin resistant by virtue of a spontaneous chromosomal mutation),and Spectinomycin/Streptomycin-resistant isolates were verified byrestriction digestion analysis to contain intact plasmid pDAB101513prior to preparation of frozen glycerol stocks and storage at −80° C.This strain is named EHA105(pDAB101513). Numerous individual culturesestablished from cells obtained from frozen glycerol stocks ofEHA105(pDAB101513) were found to contain re-arranged or deleted versionsof the pDAB101513 plasmid. For maize transformations, bulk cells ofstrain EHA105(pDAB101513) were harvested from an agar plate inoculatedfrom a frozen glycerol stock and used directly as described in Example13.

Plasmid pDAB101513 was successfully introduced by electroporation intoelectrocompetent cells of A. tumefaciens strain DA2552 (essentially aRecA-deficient version of strain EHA105) to produce strainDA2552(pDAB101513). Transformants selected by means of resistance toErythromycin and Spectinomycin were validated by restriction enzymedigestion of plasmid DNA prior to preparation of frozen glycerol stocksand storage at −80° C. Numerous individual cultures established fromcells obtained from frozen glycerol stocks were found to contain intactpDAB101513 plasmid. Bulk cells of strain DA2552(pDAB101513) wereharvested from an agar plate inoculated from a frozen glycerol stock andused for maize transformations (Example 13).

Plasmid pDAB101513 was successfully introduced by electroporation intoelectrocompetent cells of A. tumefaciens strain DAt13192 (strain DA2552harboring plasmid pDAB9292) to produce strain DAt13192(pDAB101513).Transformants selected by means of resistance to Erythromycin,Kanamycin, and Spectinomycin were validated by restriction enzymedigestion of plasmid DNA prior to preparation of frozen glycerol stocksand storage at −80° C. Numerous individual cultures established fromcells obtained from the frozen stocks were found to contain intactpDAB101513 plasmid. Bulk cells of strain DAt13192(pDAB101513) wereharvested from an agar plate inoculated from a frozen glycerol stock andused for maize transformations (see Example 13).

In similar fashion, a derivative of pSB11 (the shuttle vector of thesuperbinary system) was constructed having a T-DNA region analogous tothat of pDAB101513. Multiple attempts to construct a superbinary plasmidby standard methods in LBA4404(pSB1) were unsuccessful. All attemptsresulted in isolation of highly rearranged and deleted pSB1-basedcointegrant plasmids.

Example 12: Construction of Plant Transformation Vector pDAB101514Having Multiple Repeated Sequence Elements and Introduction intoAgrobacterium Strains

The utility of an engineered A. tumefaciens strain having a deficiencyin RecA function in combination with the auxiliary vir genes provided bythe 14.8 KpnI VirBCDG fragment is further illustrated herein. A binaryplant transformation vector, pDAB101514 (FIG. 4B), was constructed in E.coli cloning strain STBL2™ by a combination of standard cloning methodsand GATEWAY™ technology. The structure of binary vector pDAB101514 isnearly the same as that of pDAB101513 (previous Example) with theexception of the expression elements used to drive expression of thecry1Ca gene. The transcription of the cry1Ca CDS in pDAB101514 is drivenby a 1429 bp sugarcane bacilliform virus promoter (SCBV; Tzafrir et al.(1998) Plant Molec. Biol. 38:347-356). The 5′UTR is comprisedessentially of intron 6 of the maize alcohol dehydrogenase gene (GENBANKAccession X04049), flanked by twenty bases of exon 6 and eleven bases ofexon 7. The transcription of this gene is terminated by a potato pinII3′UTR (An et al. (1989) Plant Cell 1:115-122). The expression elementsused to control expression of the cry1Fa, cry1Ab, and aad1 genes are thesame as were employed in pDAB101513. Thus, within the 22,586 bp T-DNAregion of pDAB101514, the three copies of the maize ubi1 promotercomprise a total of 5,973 bases arranged in three direct repeats ofalmost 2 kbp each, with each repeat being 100% related to the other. Thetwo copies of the Per5 3′UTR comprise a total of 730 bases arranged intwo direct repeat units, each one being 100% related to the other, andthe three coding regions cry1Ca, cry1Fa, or cry1Ab are arranged asdirect repeats having between 67% and 73% DNA sequence homology to oneanother. In total, the T-region of pDAB101514 comprises about 76% highlyrepeated DNA sequences, and the physical arrangement may be convenientlyillustrated below:

-   -   RB>SCBV promoter: cry1Ca CDS:pinII 3′UTR>Ubi1 promoter: cry1Fa        CDS:Per5 3′UTR>Ubi1 promoter: cry1Ab CDS:Per5 3′UTR>Ubi1        promoter:aad1 CDS:Lip 3′UTR>LB

The highly repeated nature of this construct required that the cloningsteps be completed in the E. coli cloning strain STBL2™, which isspecially engineered to maintain the integrity of clones containing suchhighly repeated DNA sequences.

Plasmid pDAB101514 was introduced by electroporation intoelectrocompetent cells of A. tumefaciens strain EHA105 (renderedStreptomycin resistant by virtue of a spontaneous chromosomal mutation),and Spectinomycin/Streptomycin-resistant isolates were verified byrestriction digestion analysis to contain intact plasmid pDAB101514prior to preparation of frozen glycerol stocks and storage at −80° C.This strain was named EHA105(pDAB101514). Numerous individual culturesestablished from EHA105(pDAB101514) cells obtained from frozen glycerolstocks were found to contain re-arranged or deleted versions of thepDAB101514 plasmid. For maize transformations, bulk cells of strainEHA105(pDAB101514) were harvested from an agar plate inoculated from afrozen glycerol stock and used by methods disclosed in Example 13.

Plasmid pDAB101514 was successfully introduced by electroporation intoelectrocompetent cells of A. tumefaciens strain DA2552 (essentially aRecA-deficient version of strain EHA105) to produce strainDA2552(pDAB101514). Transformants selected by means of resistance toErythromycin and Spectinomycin were validated by restriction enzymedigestion of plasmid DNA prior to preparation of frozen glycerol stocksand storage at −80° C. Numerous individual cultures established fromcells obtained from frozen glycerol stocks were found to contain intactpDAB101514 plasmid. Bulk cells of strain DA2552(pDAB101514) wereharvested from an agar plate inoculated from a frozen glycerol stock andused for maize transformations by methods disclosed in Example 13.

Plasmid pDAB101514 was successfully introduced by electroporation intocells of A. tumefaciens strain DAt13192 (strain DA2552 harboring plasmidpDAB9292) to produce strain DAt13192(pDAB101514). Transformants selectedby means of resistance to Erythromycin, Kanamycin, and Spectinomycinwere validated by restriction enzyme digestion of plasmid DNA prior topreparation of frozen glycerol stocks and storage at −80° C. Numerousindividual cultures established from DAt13192(pDAB101514) cells obtainedfrom the frozen stocks were found to contain intact pDAB101514 plasmid.Bulk cells of strain DAt13192(pDAB101514) were harvested from an agarplate inoculated from a frozen glycerol stock and used for maizetransformations by methods disclosed in Example 13.

In similar fashion, a derivative of pSB11 (the shuttle vector of thesuperbinary system) was constructed having a T-DNA region analogous tothat of pDAB101514. Multiple attempts to construct a superbinary plasmidby standard methods in LBA4404(pSB1) were unsuccessful. All attemptsresulted in isolation of highly rearranged and deleted pSB1-basedcointegrant plasmids.

Example 13: Transformation of Maize by Agrobacterium Strains HarboringBinary Vectors pDAB101513 and pDAB101514

Agrobacterium-Mediated Transformation of Maize:

Seeds from a Hi-II F1 cross (Armstrong et al. (1991) Maize Genet. Coop.Newslett. 65:92-93) were planted into 5-gallon-pots containing a mixtureof 95% METRO-MIX 360 soilless growing medium (SUN GRO HORTICULTURE;Bellevue, Wash.) and 5% clay/loam soil. The plants were grown in agreenhouse using a combination of high pressure sodium and metal halidelamps with a sixteen hours light/eight hours dark photoperiod.Controlled sib-pollinations were performed to obtain immature F2 embryosfor transformation. Maize ears were harvested at approximately eight toten days post-pollination when immature embryos were between 1.0 mm and2.0 mm in size.

Infection and Co-Cultivation:

Maize ears were dehusked and surface sterilized by scrubbing with liquidsoap, immersing in 20% commercial bleach (containing 5% sodiumhypochlorite) for about 20 minutes, then rinsing three times withsterile water. A suspension of A. tumefaciens cells harboring pDAB101513or pDAB101514, binary vectors having three genes encoding the Bt Cry1Ca,Cry1Fa, and Cry1Ab proteins, and containing the aad-1 plant selectablemarker gene, was prepared by transferring one or two loops of bacteria(grown for two to three days at 28° C. on YEP agar medium containingappropriate antibiotics) into 5 mL of liquid infection medium (LS BasalMedium (Linsmaier and Skoog (1965) Physiologia Plantarum 18:100-127), N6vitamins (Chu et al. (1975) Scientia Sinica 18:659-668), 1.5 mg/L2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5 gm/L sucrose, 36.0 gm/Lglucose, 6 mM L-proline, pH 5.2] containing 200 μM acetosyringone. Thesolution was vortexed until a uniform suspension was achieved, and theconcentration was adjusted to a final optical density of approximately0.4 at 550 nm.

Immature embryos were isolated directly into a microcentrifuge tubecontaining 2 mL of the infection medium. The medium was removed andreplaced with 1 mL of the Agrobacterium solution and theAgrobacterium/embryo solution was incubated for five to ten minutes atroom temperature. Embryos were then transferred to cocultivation medium(LS Basal Medium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mML-proline, 0.85 mg/L AgNO3, 2.8 gm/L GELLAN GUM™ (PHYTOTECHNOLOGYLABORATORIES; Lenexa, Kans.), pH 5.8) containing 200 μM acetosyringoneand cocultivated for three to four days at 20° C. in the dark.

After cocultivation, the embryos were transferred to resting mediumcontaining MS salts and vitamins (Frame et al., 2011, GeneticTransformation Using Maize Immature Zygotic Embryos. in Plant EmbryoCulture Methods and Protocols: Methods in Molecular Biology, T. A.Thorpe and E. C. Yeung (Eds), SPRINGER SCIENCE AND BUSINESS MEDIA, LLC.pp 327-341), 6 mM L-proline, 100 mg/L myo-inositol, 500 mg/L MES(2-(N-morpholino) ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIESLABR.), 30 gm/L sucrose, 1.5 mg/L 2,4-D, 0.85 AgNO3, 250 mg/LCefotaxime, 2.8 gm/L GELLAN GUM™, pH 5.8. Approximately seven dayslater, embryos were transferred to the same medium supplemented with 100nM Haloxyfop. Transformed isolates were identified after approximatelyeight weeks and were bulked up by transferring to fresh selection mediumat two-week intervals for regeneration and analysis.

Regeneration and Seed Production:

For regeneration, the cultures were transferred to “28” induction medium(MS salts and vitamins, 30 gm/L sucrose, 5 mg/L Benzylaminopurine, 0.25mg/L 2, 4-D, 250 mg/L Cefotaxime, 2.5 gm/L GELLAN GUM™, pH 5.7)supplemented with 100 nM Haloxyfop. Incubation was for one week underlow-light conditions (14 μEm⁻² s⁻¹), then one week under high-lightconditions (approximately 89 μEm⁻² s⁻¹). Tissues were subsequentlytransferred to “36” regeneration medium (same as induction medium exceptlacking plant growth regulators). When plantlets were 3 cm to 5 cm inlength, they were transferred to glass culture tubes containing SHGAmedium [(Schenk and Hildebrandt salts and vitamins, PHYTOTECHNOLOGIESLABR.), 1.0 gm/L myo-inositol, 10 gm/L sucrose and 2.0 gm/L GELLAN GUM™,pH 5.8] to allow for further growth and development of the shoot androots. Plants were transplanted to the same soil mixture as describedearlier and grown to flowering in the greenhouse. Samples of planttissues were harvested and used in insect bioassays by methods disclosedin Example 14 and for molecular and biochemical analyses. Controlledpollinations for seed production are conducted.

Those skilled in the art of maize transformation will understand thatother methods are available for maize transformation and for selectionof transformed plants when other plant expressible selectable markergenes (e.g., herbicide tolerance genes) are used.

Example 14: In Vitro Bioassays of Leaf Samples Against Maize InsectPests

The lepidopteran species assayed were the corn earworm (CEW; Helicoverpazea (Boddie)), European corn borer (ECB; Ostrinia nubilalis (Hübner)),and fall armyworm (FAW; Spodoptera frugiperda (J. E. Smith)). Eggs forthese insects were obtained from BENZON RESEARCH (Carlisle, Pa.).

First Tier Bioassay: High-Throughput 96-Well Bioassay:

96-well trays (TPP-US; St. Louis, Mo.) were partially filled with a 2%agar solution (SIGMA-ALDRICH) and agar was allowed to solidify. Using astandard hand-held paper punch, three ⅛ inch diameter leaf discs weresampled for each of the two insect species (CEW and FAW) tested in thisformat. One leaf disc was placed in a single well of the 96-well plate;there were three plates for each insect tested (one for eachreplicate/leaf disc). An egg-seeding device was used to administerinsect eggs into each well of the 96-well plate. Plates were then sealedwith perforated sticky lids and also enclosed with the plastic lid thataccompanies the plates. Plates were held at 30° C., 40% RelativeHumidity (RH), sixteen hours light/eight hours dark for three days.Grading was conducted using a 0-1-2 scale, in which 0 indicated <25%leaf disc damage, 1 indicated 25-50% leaf disc damage, and 2indicated >50% leaf disc damage within each well. Damage scores for eachtest were averaged and used alongside protein expression analysis toconduct correlation analyses. Plants whose average insect damage scorewas 0.67 or less were considered active against the tested pest.

Second Tier Bioassay: 32-Well Bioassay:

32-well trays (C-D INTERNATIONAL; Pitman, N.J.) were partially filledwith a 2% agar solution and agar was allowed to solidify. Leaf sectionsapproximately 1 inch square were taken from each plant and placed singlyinto wells of the 32-well trays. One leaf piece was placed into eachwell, and two leaf pieces were tested per plant and per insect. Insects(ECB and FAW) were mass-infested using a paintbrush, placing ten totwenty neonate larvae into each well. Trays were sealed with perforatedsticky lids which allowed ventilation during the test. Trays were placedat 28° C., 40% RH, sixteen hours light/eight hours dark for three days.After the duration of the test, a simple percent damage score was takenfor each leaf piece. Damage scores for each test were averaged and usedalongside protein expression analysis to conduct correlation analyses.Plants whose average insect damage ratings were 25% or less wereconsidered active against the tested pest.

Statistical Analysis:

All analyses were conducted in JMP 8.0.2 (SAS INSTITUTE Inc., Cary,N.C.). One-way ANOVA analysis was used to determine significantdifferences between the treatments and the negative control plants forinsect damage data. The Tukey-Kramer HSD comparison of means was alsoused to further evaluate significant differences among the treatments.In addition, linear regression (least fit squares) analysis was used tocorrelate quantitative protein expression with insect activitymeasurements.

Bioassay results are summarized in Table 2.

Example 15: Biochemical and Molecular Characterization of Maize TissuesTransformed with pDAB101513

Multiple transformation experiments were performed with engineered A.tumefaciens strains EHA105(pDAB101513), DA2552(pDAB101513) andDAt13192(pDAB101513). Copy numbers of the four transgenes in transgenicT₀ plants were estimated by hydrolysis probe assays (Bubner and Baldwin(2004) Plant Cell Rep. 23:263-271) using gene-specific oligonucleotides.Protein extracts from plants with one to three copies (“Low Copy”) ofthe genes were further examined for production of the Bt Cry1Ca, Cry1Fa,and Cry1Ab proteins, and for the AAD1 protein, by ELISA methods usingcommercially produced antibody kits (ENVIROLOGIX™, Portland, Mass.).Some plants were found that produced all four proteins (Table 3). Inaddition, leaf pieces from the plants were bioassayed for activityagainst three maize insect pests: corn earworm (CEW, Helicoverpa zea),fall armyworm (FAW, Spodoptera frugiperda) and European corn borer (ECB,Ostrinia nubilalis) in feeding assays (EXAMPLE 14). Some plants werefound that had all four transgenes in low copy number, produced all fourproteins, and had insect activity against all three pests (Table 4). Notransformed plants meeting these criteria were obtained from experimentsusing the EHA105(pDAB101513) or DA2552(pDAB101513) strains (Table 4).Thus, a feature of strain DAt13192, comprising a deletion of thechromosomal recA gene, further comprising a full set of pTiBo542-derivedvir genes harbored on pTiEHA105, and even further comprising a partialset of pTiBo542-derived vir genes harbored on the 14.8 KpnI VirBCDGfragment of pDAB9292, is that it is able to efficiently producetransformed maize plants with large T-DNA regions comprised of highlyrepeated sequence elements.

Example 16: Biochemical and Molecular Characterization of Maize TissuesTransformed with pDAB101514

Multiple transformation experiments were performed with engineered A.tumefaciens strains EHA105(pDAB101514), DA2552(pDAB101514), andDAt13192(pDAB101514). Copy numbers of the four transgenes in transgenicT₀ plants were estimated by hydrolysis probe assays (Bubner and Baldwin,supra) using gene-specific oligonucleotides. Protein extracts fromplants with one to three copies (“Low Copy”) of the genes were furtherexamined for production of the Bt Cry1Ca, Cry1Fa, and Cry1Ab proteins,and for the AAD1 protein, by ELISA methods using commercially producedantibody kits (ENVIROLOGIX™, Portland, Mass.). In addition, leaf piecesfrom the plants were bioassayed for activity against three maize insectpests in feeding assays (Example 14). Some plants were found that hadall four transgenes in low copy number, produced all four proteins, andhad insect activity against all three pests (Table 5). No transformedplants meeting these criteria were obtained from experiments using theEHA105(pDAB101514) or DA2552(pDAB101514) strains. Thus, a feature ofstrain DAt13192, comprising a deletion of the chromosomal recA gene,further comprising a full set of pTiBo542-derived vir genes harbored onpTiEHA105, and even further comprising a partial set of pTiBo542-derivedvir genes harbored on the 14.8 KpnI VirBCDG fragment of pDAB9292, isthat it is able to efficiently produce transformed maize plants withlarge T-DNA regions comprised of highly repeated sequence elements.

Example 17: Identification and Characterization of a Neutral IntegrationSite in the Agrobacterium tumefaciens LBA4404 Chromosome

The plant-inducible picA/pgl locus of the A. tumefaciens strain C58chromosome (GENBANK Accession AE0009243) was identified as anon-essential gene into which DNA fragments could be integrated (Rong etal. (1990) J. Bacteriol. 172:5828-5836; Rong et al. (1991) J. Bacteriol.173:5110-5120). A similar neutral integration site in the genome of A.tumefaciens strain LBA4404 has not been reported. We describe here theidentification and sequencing of a genomic region of LBA4404 thatincludes sequences partially homologous to the C58 picA/pgl locus. Cellsof LBA4404 (INVITROGEN) were grown in YM medium (gm/L: yeast extract,0.4; mannitol, 10; NaCl, 0.1; MgSO₄ 7H₂O, 0.2; K₂HPO₄ 3H₂O, 0.5) at 30°C. overnight. Genomic DNA was prepared from a 1 mL culture using theEASY DNA kit (INVITROGEN) according to the manufacturer's protocols.Degenerate primers were designed based upon two regions of homologybetween the C58 PicA protein sequence and homologues identified fromArabidopsis thaliana, Caldicellulosiruptor saccharolyticus, Alkahphilusmetalliredigenes, and Clostridium acetobutylicum. LBA4404 genomic DNAwas used as a template for the polymerase chain reaction (PCR) usingHERCULASE™ MASTER MIX (STRATAGENE; San Diego, Calif.) and degenerateprimers AtnilA1Fa (5′-GACAGTCCNAATACSGAYGG-3′; SEQ ID NO:14;corresponding to amino acids 273-279 of the C58 PicA protein) andAtnilA3R (5′-GTYTTSAGNCGSAGSCCSCGRTCSGT-3; SEQ ID NO:15, correspondingto the complementary strand coding for amino acids 364-369 of the C58PicA protein). Thermocycling conditions used were: one cycle of 94° C.,2 minutes; 25 cycles of (94° C., 30 seconds; 55° C., 30 seconds; 72° C.,60 seconds); one cycle of 72° C., 7 minutes. Degenerate nucleotidedesignations in the primer sequences correspond to DNA nucleotides asfollows: N=A, C, G, or T; Y=T or C; R=A or G; and S=C or G. A 285 basepair (bp) product was isolated, cloned into the vector pCR2.1-TOPO(INVITROGEN) in Escherichia coli TOP10 cells (INVITROGEN), and the DNAsequence was determined. The sequence was found to be homologous, butnot identical, to a region of the C58 picA gene (85% sequence identity),and the LBA4404 genomic region that it represents is referred to hereinas the nilA fragment.

Additional primers complementary to the 285 bp LBA4404 nilA fragmentwere designed to be used as anchors for PCR amplification of genomicfragments flanking both ends of the 285 bp sequence. These were pairedin the PCR reactions with primers designed from sequences of theflanking regions of the C58 picA gene. Sequences of amplified fragmentsoriginating from within the 285 bp sequence and extending into both nilAfragment flanking regions were determined and used to design otherprimers for subsequent PCR reactions. Using LBA4404 genomic DNA templatewith primers nilA2F (5′-CCATCCTCATAACACCAGCT-3; SEQ ID NO:16) and nilA2R(5′-GCAGATCATCGATACGACCA-3; SEQ ID NO:17), an approximately 2 kilobase(kbp) PCR fragment was generated and cloned into pCR®-BLUNT II/XL-TOPO®using the TOPO TA cloning kit (INVITROGEN) to produce plasmid pDOW3719(FIG. 2). Sequence analysis of the insert fragment of pDOW3719 yieldedan 1,796 bp sequence (SEQ ID NO:18) which comprises a longest openreading frame (ORF) that encodes a putative protein of 531 amino acids.A shorter ORF in the same reading frame encodes a putative protein of523 amino acids. The LBA4404 523 amino acid putative protein shows 88%similarity, 85% identity with the C58 PicA protein. The coding sequencesfor the LBA4404 523 amino acid putative protein and the C58 PicA proteinhave 81% identity. Thus, the nilA fragment of LBA4404 represents agenomic segment that includes a putative gene that is substantiallydiverged from the C58 picA gene. In this disclosure, the 1.8-kbp genomicsequence represented by SEQ ID NO:18 is referred to as the nilA locus.

Plasmid pDOW3719, having the colE1 origin of replication, is notexpected to replicate autonomously in A. tumefaciens cells. DNA ofplasmid pDOW3719 was used to transform cells of A. tumefaciens strainLBA4404 by electroporation. Selection for Kanamycin resistance (harboredon pDOW3719) identified transformants that had integrated pDOW3719 intothe chromosome of LBA4404 via recombination mediated by the 1.8-kbphomology regions present in the LBA4404 chromosome and on pDOW3719. Suchan integration event results in the creation of a linear copy of thepDOW3719 vector plasmid sequence flanked on each side by thenow-duplicated 1.8-kbp homology region. Kanamycin resistant LBA4404transformants were isolated and screened for insertion of pDOW3719 byPCR analysis. Genomic DNA preparations of the transformants were used astemplate in PCR reactions with five primers sets: i) M13F primer pairedwith M13R primer, which flank the insert in pDOW3719, ii) M13F primerpaired with primer AS4R (comprising bases complementary to residues 1041to 1060 of SEQ ID NO:18), iii) M13F primer paired with primer AS10R(comprising bases complementary to residues 1,320 to 1,337 of SEQ IDNO:18), iv) M13F primer paired with primer AS11R (comprising basescomplementary to residues 1,391 to 1,406 of SEQ ID NO:18), and v) M13Rprimer paired with primer AS9F (comprising bases 634 to 649 of SEQ IDNO:18). In control reactions, all of these primer sets amplifiedexpected sized fragments when pDOW3719 plasmid DNA was used as template.However, when genomic DNA from a Kanamycin resistant LBA4404transformant was used as template, PCR using the M13F and M13R primerpair did not yield amplified products, indicating that no intact(non-integrated) pDOW3719 plasmid DNA was co-purified with the genomicDNA. PCR analysis of the genomic DNA samples with the other four primerpairs showed production of expected sized DNA fragments. These resultsindicate that the Kanamycin resistance of the LBA4404 transformants isconferred by pDOW3719 DNA which has integrated into the genome.

One such transformant [LBA4404nilA-int1] was used to test the effectthat the genomic insertion into the nilA locus has on the ability of thestrain to transform Arabidopsis thaliana. Binary vector pDAB3779, whichcontains a plant expressible gene encoding the PAT protein (whichconfers resistance to the herbicide BASTA™) was transformed into cellsof strains LBA4404nilA-int1 and LBA4404, with selection forSpectinomycin resistance. These strains were then used to conductArabidopsis transformation experiments using the methods of Weigel andGlazebrook (supra). No difference was seen in the transformationfrequencies obtained with the two strains. Thus, a feature of theembodiments and methods described herein is that insertion of a foreignDNA fragment into the chromosomal nilA locus of A. tumefaciens strainLBA4404 that comprises SEQ ID NO:18 has no effect on the growth or planttransformation capability of such engineered strain.

Example 18: Construction of a Suicide Derivative of pDOW3719 forIntegration into the LBA4404 nilA Locus

The insertion of a multiple cloning site in the nilA locus cloned inpDOW3719 was accomplished by splice overlap extension (SOE) PCR (Hortonet al. (1990) BioTechniques 8:528-535). SOE PCR reactions were carriedout using HERCULASE™ master mix according to the manufacturer'sprotocols. A portion of the nilA locus was amplified using pDOW3719 DNAas template with primer nilA5′(5′-CCGGCTCTTCCAGCTCCTCATGCACGAACAACGAGAAACGAGC-3; SEQ ID NO:19) pairedwith primer nilA_MCS_SOER (5′-GAATGGTGAAACCTCTAGATTAATTAAGGATCCCCGGGTACCGAAAAGCCCGACATTGC-3′; SEQ ID NO:20) to produce anapproximately 800 bp fragment. A second portion of the nilA locus wasamplified using pDOW3719 DNA as template and primer nilA_MCS_SOEF(5′-GCAATGTCGGGCTTTTCGG TACCCGGGGATCCTTAATTAATCTAGAGGTTTCACCATTC-3′; SEQID NO:21) paired with primer nilA3′(5′-GGAATTCTCAGTGGCTTTCATGGGTTTTCTCG-3′; SEQ ID NO:22) to produce anapproximately 900 bp fragment. The resulting fragments were then gelpurified (NUCLEOSPIN™, CLONTECH; Mountain View, Calif.), and used astemplate for amplification with primers nilA5′ and nilA3′ to yield a1.6-kbp fragment, which sequence is disclosed as SEQ ID NO:23 (nilAMCS). The resultant fragment was digested with Pvu I and Sap I (NEB) andligated to pBCSK+sacBI DNA (INVITROGEN) digested with the samerestriction enzymes, using T4 DNA ligase (NEB). E. coli TOP10 cells weretransformed with the ligation mixture, and transformants were selectedon LB soy agar (TEKNOVA; Hollister, Calif.) supplemented with 30 μg/mLChloramphenicol. Clones were screened by restriction digestion with PvuI and Kpn I (NEB). The nilA locus region of positive clones was sequenceverified, and the resulting plasmid was named pDOW3721. A feature ofpDOW3721 is that a multiple cloning site (MCS) containing recognitionsequences for restriction enzymes Sph I, Kpn I, Sma I, BamH I, Pac I,Ase I and Xba I is flanked on one side by 852 bp of LBA4404-derivedbases, and on the other side by 745 bp of LBA4404-derived bases.

Thus, a foreign DNA fragment may be cloned into the MCS of pDOW3721, andthence integrated into the LBA4404 chromosomal nilA locus by virtue ofhomologous recombination mediated by the LBA4404-derived flankingsequences. Single crossover events, by means of which the entirepDOW3721 plasmid sequence is integrated into the LBA4404 chromosome, maybe resolved into double crossover events by counterselection on sucrosecontaining media. On such media, the sucrose is converted to a toxicproduct upon enzymolysis by the SacB protein encoded by the sacB gene(Reid and Collmer (1987) Gene 57:239-246; Quandt et al. (1993) Gene127:15-21). Thus, transformants able to survive on sucrose-containinggrowth medium will have undergone a second crossover event thateliminates the pDOW3721 plasmid vector backbone from the chromosome,leaving behind the disrupted nilA locus containing the integratedforeign DNA fragment. Many reports have shown for the last ten yearsthat the transfer of vector backbone sequences is quite common. Theratio of the plants that acquired the backbone sequences intransformants ranged typically between 20% and 50%, and was sometimes ashigh as 75% or more.

As one exemplification of the utility of pDOW3721, the 14.8 KpnI VirBCDGfragment was prepared from plasmid pSB1 and ligated to Kpn I digestedpDOW3721 DNA, using T4 DNA ligase. E. coli TOP10 cells were transformedwith the ligation mixture, and transformants were selected on LB soyagar supplemented with 30 μg/mL Chloramphenicol. Clones were screened byrestriction digestion with EcoR I and Hind III. The resultant plasmidwas named pDOW3722.

Example 19: Identification of Nucleotide Sequences Upstream andDownstream of nilA

The LBA4404nilA-int1 strain of A. tumefaciens, containing a genomicintegration of plasmid pDOW3719 (FIG. 2 and Example 18), was used toidentify additional sequences positioned upstream and downstream of thenilA genomic region. pDOW3719 contains a 1,796 bp PCR amplicon of the A.tumefaciens strain LBA4404 nilA locus cloned into PCR-BLUNT II/XL-TOPO®(INVITROGEN). This plasmid was integrated into the genome of A.tumefaciens strain LBA4404 via homologous recombination. Colonies whichcontained the integrated plasmid were identified by resistance toKanamycin (Example 17). The integrated plasmid, and the elementscontained within it, may be used as tools for the isolation andcharacterization of additional nucleotide sequences via a “plasmidrescue” technique.

Genomic DNA (gDNA) was prepared from cells of the LBA4404nilA-int1strain by a protocol for bacterial genomic DNA isolation (Sambrook etal., supra). One microgram of gDNA was individually digested with thefollowing enzymes (all obtained from NEB): Hind III, BamH I, Pst I, AscI, and Sac II. These restriction enzymes were chosen specifically toproduce gDNA fragments that map upstream and downstream of the nilAlocus. The Hind III, BamHI, and Pst I restriction enzymes were selectedbecause their recognition sites are unique within the pDOW3719 sequence.Moreover, these enzyme recognition sites are located at the junctionsbetween the nilA locus amplicon fragment and the PCR-BLUNT II/XL-TOPO®vector (FIG. 2). Cleavage of gDNA with these enzymes and self ligationof the resulting fragments thus results in a plasmid rescue fragmentwhich contains the uncharacterized genomic sequences ligated adjacent tothe M13 forward universal primer or the M13 reverse universal primerbinding sites of the pDOW3917 plasmid. Such clones are isolated bytransforming the ligation mixture into E. coli cells, with selection forthe Kanamycin resistance gene harbored by pDOW3917.

Further, the pDOW3719 plasmid does not contain recognition sites for theAsc I and Sac II restriction enzymes. Therefore, gDNA fragmentsgenerated by these restriction enzymes would produce a chimeric DNAfragment which spans the entire length of the integrated pDOW3719plasmid sequence and includes the gDNA regions which flank both sides ofthe integrated pDOW3719 plasmid.

The gDNA fragments which resulted from restriction enzyme digestion asdescribed above were self-ligated using T4 Ligase (ROCHE APPLIEDSCIENCES; Indianapolis, Ind.). The ligation products were transformedinto E. coli ONESHOT® TOP10 CELLS (INVITROGEN) and plated on LB mediacontaining Kanamycin (50 μg/mL). Individual colonies were selected andplasmid DNA was isolated and characterized via plasmid restrictionenzyme digestion patterns. Clones which contained plasmids exhibiting aconsistent restriction enzyme digestion banding pattern as compared toone another were advanced for use in sequencing reactions.

The nucleotide sequences of the gDNA upstream and downstream from thenilA locus were determined using a “genome walking” technique.Sequencing primers corresponding to the known gDNA sequence (as presentin pDOW3719) were designed and used with the CEQ™ DYE TERMINATOR CYCLESEQUENCING KIT according to the manufacturer's recommendations (BECKMANCOULTER; Fullerton, Calif.). From the determined sequence, a second setof primers, located in previously unknown genomic sequence, was designedand used to generate additional sequencing data. This process wasrepeated until all of the available gDNA nucleotide sequence wasdetermined. This technique generated 2,936 bp of sequence upstream fromthe nilA locus, and 4,361 bp of sequence downstream from the nilA locus.In combination with the 1,796 bp of the previously identified nilAlocus, the newly identified upstream and downstream flanking sequencesregions netted a 9,093 bp sequence comprising the nilA genomic region(SEQ ID NO:24), which extends in both directions from the originallyidentified nilA locus.

Example 20: Construction of a Vector for Integration into the LBA4404nilA Genomic Region

An integration vector for homology-mediated integration of foreign DNAsequences into the A. tumefaciens LBA4404 nilA genomic region wasdesigned and constructed. A 6.3-kbp fragment of the nilA genomic regionspanning the nilA locus was PCR amplified using the FAILSAFE™ PCR KIT(EPICENTRE®, Madison, Wis.). The amplified fragment was ligated into thePCR® 8/GW/TOPO® vector (INVITROGEN), and positive clones were confirmedvia restriction enzyme digestion and DNA sequence verification. Theresulting vector, pDAB9615, was further modified by the addition of anoligonucleotide fragment containing multiple unique restriction enzymerecognition sites. These restriction sites, flanked by 3,244 bp and3,128 bp regions of the nilA genomic region, serve as cloning sites forthe introduction of foreign nucleotide sequences. The resulting vectorwas named pDAB9618 (FIG. 3).

A 15,549 bp fragment containing the 14.8 KpnI VirBCDG fragment of pSB1and a bacterial Kanamycin resistance gene was prepared by digestion ofpDAB9292 DNA with Kpn I plus Bstl107I. This fragment was then ligated toDNA of pDAB9618 that had been digested with Kpn I plus Swa I, to producevector pDAB9621 (FIG. 3). The GATEWAY® reaction was then used to movethe portion of pDAB9621 containing the 14.8 KpnI VirBCDG fragment andbacterial Kanamycin resistance gene, flanked on each side by 3 kbp ofLBA4404 nilA genomic region sequences, into the GATEWAY® PDEST™14 vectorvia AN L-R CLONASE® reaction (INVITROGEN). The resulting plasmid,pDAB9698 (FIG. 3, SEQ ID NO:25), was confirmed via restriction enzymedigestion and DNA sequencing reactions. pDAB9698 served as anintegration vector for integrating the pTiBo542-derived vir genes frompSB1 (harbored on the 14.8 KpnI VirBCDG fragment) into the nilAchromosomal region of A. tumefaciens strain LBA4404.

Example 21: Chromosomal Integration of the 14.8 KpnI VirBCDG FragmentVia Homologous Recombination

DNA of plasmid pDAB9698 was produced using a NUCLEOBOND® AX ANIONEXCHANGE CHROMATOGRAPHY PLASMID DNA ISOLATION KIT (MACHEREY-NAGEL). Thepurified plasmid DNA was electroporated into A. tumefaciens LBA4404CELLS (INVITROGEN). Briefly, 500 ng of plasmid DNA was incubated withthe cells at 4° C. for ten minutes. This mixture was pipetted into anice-chilled 0.2 cm GENE PULSER® CUVETTE (BIO-RAD) and electroporatedusing the BIO-RAD GENE PULSER with the following settings: capacitanceoutput 25 μFarad, capacitance extender 960 μFarad, resistance 200 ohms,and voltage 2.5 kVolts. Immediately after electroporation, 950 μL of SOCmedium (INVITROGEN) was added and the mixture was transferred to aFalcon 2059 tube (BECTON DICKINSON AND CO.; Franklin Lakes, N.J.). Thetransformed cells were then incubated at 28° C. for five to six hours.After incubation, the cells were plated on separate YEP medium platescontaining Kanamycin (50 μg/mL). The plates were grown inverted at 28°C. for 36 to 48 hours. Single colonies were picked and propagated in 5mL of liquid YEP containing Kanamycin (50 μg/mL) for approximately 36hours at 28° C. These cultures were used to prepare glycerol stockcultures by vigorous mixing with an equal volume of 100% sterileglycerol, followed by freezing and storage at −80° C.

Example 22: Phenotypic and Molecular Confirmation of the ChromosomalIntegration of the 14.8 KpnI VirBCDG Fragment

pDAB9698, having the colE1 origin of replication, is not expected toreplicate autonomously in A. tumefaciens cells. Thus, upontransformation of pDAB9698 DNA into LBA4404 cells, stable Kanamycinresistance results from the integration of DNA of pDAB9698 intoautonomously replicating Agrobacterium genetic elements. These plasmidintegrants will fall into four classes that can be used according tovarious embodiments of the Agrobacterium strains and methods for theiruse as described herein. The first class comprises cells in whichpDAB9698 DNA has integrated into a site remote from the nilA genomicregion by means of nonhomologous recombination. These cells should beKanamycin resistant by virtue of the Kanamycin resistance gene adjacentto the 14.8 KpnI VirBCDG fragment, and additionally should be resistantto Ampicillin by virtue of the Ampicillin resistance gene harbored onthe pDAB9698 backbone vector (pDEST™14). The second class comprisescells in which the pDAB9698 DNA has integrated into the autonomouslyreplicating pAL4404 Ti helper plasmid (natively resident in LBA4404) byvirtue of homologous recombination mediated by the pTiBo542-derivedVirBCDG genes present on pDAB9698 and the pTiACH5-derived VirBCDG genespresent on pAL4404. These cells should also be resistant to bothKanamycin and Ampicillin. The third class comprises cells in whichpDAB9698 DNA has integrated into the LBA4404 nilA genomic region byvirtue of a single homologous recombination (crossover) event mediatedby either of the approximately 3-kbp nilA genomic region sequencesharbored on pDAB9698, and which flank the 15,549 bp fragment containingthe 14.8 KpnI VirBCDG fragment of pSB1 and the Kanamycin resistancegene. These cells should also be resistant to both Kanamycin andAmpicillin. The fourth class comprises cells in which the singlecrossover event of class 3 cells above undergoes a second crossoverevent mediated by the now-duplicated 3-kbp nilA genomic region sequencesthat are generated as a consequence of the single crossover event.Depending upon which of the flanking 3-kbp nilA genomic region sequencesgenerated the single crossover event, and which of these flankingsequences generates the second crossover event, the resultant cellsshould either be Kanamycin sensitive, and Ampicillin resistant, orKanamycin resistant and Ampicillin sensitive. Preferred cells asdescribed herein comprise the latter class, that is, cells that areKanamycin resistant and Ampicillin sensitive. These double crossoverevents, which do not contain the pDEST™14 plasmid backbone, aredesirable as they do not contain superfluous genetic elements such asthe colE1 replication origin and Ampicillin resistance gene.

Putative transformants isolated in Example 21 were screened for adesirable double homologous recombination-mediated integration event.Kanamycin-resistant isolates having the 15,549 bp fragment containingthe 14.8 KpnI VirBCDG fragment of pSB1 and the Kanamycin resistance gene(and lacking the pDEST™14 vector backbone) were identified viasensitivity to Ampicillin. The putative transformants were grown in 3 mLof YEP containing Kanamycin (50 μg/mL) at 28° C. for approximately 36hours. These cultures were then streaked onto solid YEP media containingvarious single antibiotics as follows (concentrations in μg/mL):Rifampicin, 100; Kanamycin, 50; Streptomycin, 125; Chloramphenicol, 50;Erythromycin, 200; Tetracycline, 12.5; and Ampicillin, 100. The plateswere incubated at 28° C. for 48 hours and colony growth was scored. Astrain was identified that was resistant to Kanamycin, Rifampcin(chromosomal marker), and Streptomycin (pAL4404 marker; Ooms et al.,supra). Moreover, the strain was sensitive to Chloramphenicol,Erythromycin, and Tetracycline. Most significantly, the strain wassensitive to Ampicillin. This drug screen phenotype is indicative of adesirable double crossover homologous recombination event, wherein the15,549 bp fragment containing the 14.8 KpnI VirBCDG fragment of pSB1 andthe Kanamycin resistance gene are integrated into the A. tumefaciensLBA4404 chromosome. This strain is called DAt16174.

The presence of the pTiBo542-derived VirBCDG genes in strain DAt16174was further confirmed by molecular characterization. Genomic DNA ofstrain DAt16174 was isolated using a bacterial genomic DNA isolationprotocol (Sambrook et al., supra and updates thereof). PCR primers weredesigned to amplify overlapping fragments of the chromosomallyintegrated VirBCDG genes. PCR reactions using the primers described inTable 6 were completed using the FAILSAFE™ PCR KIT (EPICENTRE®) per themanufacturer's directions. Due to the large total molecular size of theintegrated VirBCDG genes, the amplifications were done to produce fiveoverlapping fragments. Amplicons of the expected size were purified fromagarose gels using the QIAEX II GEL EXTRACTION KIT (QIAGEN; Valencia,Calif.) according to the manufacturer's protocol. These fragments werecloned into the PCR2.1®-TOPO® TA vector using the PCR2.1®-TOPO® TACLONING® KIT (INVITROGEN). Bacterial colonies suspected to containclones of the PCR amplicons were confirmed via restriction enzymedigestion. The DNA sequences of the amplicon fragments were determinedusing the CEQ™ DYE TERMINATOR CYCLE SEQUENCING KIT according to themanufacturer's instructions, and the sequencing data were analyzed usingSEQUENCHER™ version 4.1.4 software (GENE CODES CORP.; Ann Arbor, Mich.).The resulting sequences produced a 22-kbp contiguous sequence whichspanned the entire 15,549 bp fragment containing the 14.8 KpnI VirBCDGfragment of pSB1 and the Kanamycin resistance gene, plus both of theapproximately 3-kbp flanking nilA genomic regions, and extended furtherinto the upstream and downstream nilA genomic regions (thereby includingLBA4404 chromosomal sequence which was not originally contained inpDAB9698).

The Agrobacterium tumefaciens identity of strain DAt16174 was verifiedvia the ketolactose test. Putatively transformed colonies were streakedout on lactose agar and incubated at 28° C. for 48 hours. The plateswere then flooded with Benedict's Solution and monitored at roomtemperature. Isolates which turned the Benedict's Solution andunderlying agar from blue to yellow were thus confirmed to beAgrobacterium.

A feature of A. tumefaciens strain DAt16174 is that it may beadvantageously used as a plant transformation agent for the transfer ofT-DNA genes from binary vectors having replication origins of, forexample, the IncP, IncW, or VS1 classes. In broad terms, the introducedbinary vector may have a replication origin of any class capable ofreplication in Agrobacterium while being compatible with the pTi originof replication (and associated functions) of the pAL4404 plasmidresident in DAt16174. Thus, it is within the range of possible uses ofstrain DAt16174 that more than one binary vector plasmid may beco-resident in strain DAt16174 if the plasmids have compatiblereplication origins (i.e., are of different incompatibility groups).Selection for such introduced binary vectors should not rely onbacterial selectable marker genes conferring either Kanamycin,Rifampicin, or Streptomycin resistance, as the DAt16174 strain isresistant to these three antibiotics.

Binary vectors can replicate autonomously in both E. coli andAgrobacterium cells. They comprise sequences, framed by the right andleft T-DNA border repeat regions, that may include a selectable markergene functional for the selection of transformed plant cells, a cloninglinker, cloning polylinker, or other sequence which can function as anintroduction site for genes destined for plant cell transformation. Theycan be transformed directly into Agrobacterium cells by electroporation,by chemically mediated direct DNA transformation, introduced bybacterial conjugation, or by other methodologies. The Agrobacterium usedas host cell harbors at least one plasmid carrying a vir region. The virregion is necessary to provide Vir proteins to perform all the requisitefunctions involved in the transfer of the T-DNA into the plant cell. Theplasmid carrying the vir region is commonly a mutated Ti or Ri plasmid(helper plasmid) from which the T-DNA region, including the right andleft T-DNA border repeats, have been deleted. Examples of Agrobacteriumstrains that contain helper plasmids and are useful for planttransformation, include, for example, LBA4404, GV3101(pMP90),GV3101(pMP90RK), GV2260, GV3850, EHA101, EHA105, and AGL1. Numerousexamples of binary vector systems are reviewed by Hellens et al. (2000,Trends Plant Sci. 5:446-451).

Additionally, the plant transformation advantages conferred upon strainLBA4404(pSB1) (used in the superbinary system) by the pTiBo542-derivedvirB operon (which includes the genes virB1, virB2, virB3, virB4, virB5,virB6, virB7, virB8, virB9, virB10, and virB11), the virG gene, the virCoperon (which comprises genes virC1 and virC2) and the part of the virDoperon comprising gene virD1, as harbored on the pSB1 plasmid, areretained in strain DAt16174. Because the superbinary vir genes listedabove are integrated into the LBA4404 chromosome, strain DAt16174 isreferred to as a SUPERCHROME strain. In contrast to the superbinarysystem, use of strain DAt16174 does not require the formation ofunstable superbinary plasmids via homologous recombination between pSB1and shuttle vectors such as pSB11. A further benefit of the SUPERCHROMEstrain is that standard binary vectors may be introduced into the strainfor plant transformation.

Example 23: Biochemical and Molecular Characterization of Maize TissuesTransformed with Various Agrobacterium Strains Harboring pDAB101556

A binary plant transformation vector, pDAB101556 (FIG. 5), wasconstructed by a combination of standard cloning methods and GATEWAY™technology. Binary vector pDAB101556 is based on the IncP-typereplication origin of plasmid RK2, and the vector backbone harbors abacterial gene conferring resistance to Spectinomycin at 100 μg/mL. TheT-DNA border repeats are derived from the TL region of pTi15955. Withinthe Right Border (RB) and multiple Left Borders (LB) of the T-DNA regionof plasmid pDAB101556 are positioned two plant-expressible proteincoding sequences (CDS). The first gene (selectable marker) comprises thecoding region for the AAD1 selectable marker protein (SEQ ID NO:13)(U.S. Pat. No. 7,838,733), which is under the transcriptional control ofa 1,991 bp maize ubiquitin) promoter with associated intron1 (U.S. Pat.No. 5,510,474). This gene is terminated by a maize Lipase 3′UTR (U.S.Pat. No. 7,179,902). The second gene (screenable marker) comprises a CDSfor a yellow fluorescent protein (YFP, essentially as disclosed in U.S.Pat. No. 7,951,923) transcription of which is controlled by a maizeubiquitin 1 promoter with associated intron 1. This gene is terminatedby a maize Per5 3′UTR (U.S. Pat. No. 6,384,207).

Plasmid pDAB101556 was successfully introduced by electroporation intocells of A. tumefaciens strain LBA4404 to produce strain LBA4404(pDAB101556). This strain/plasmid combination thus comprises a standardbinary plant transformation system. Transformants selected by means ofresistance to Streptomycin and Spectinomycin were validated byrestriction enzyme digestion of plasmid DNA prior to preparation offrozen glycerol stocks and −80° C. storage. Bulk cells of strainLBA4404(pDAB101556) were harvested from an agar plate inoculated from afrozen glycerol stock and used for maize transformations by methodsdisclosed in Example 24.

Plasmid pDAB101556 was successfully introduced by electroporation intocells of A. tumefaciens strain DAt13192 (see Example 7) to producestrain DAt13192(pDAB101556). This strain/plasmid combination thuscomprises a recombination-deficient ternary plant transformation system.Transformants selected by means of resistance to Erythromycin,Kanamycin, and Spectinomycin were validated by restriction enzymedigestion of plasmid DNA prior to preparation of frozen glycerol stocksand storage at −80° C. Bulk cells of strain DAt13192(pDAB101556) wereharvested from an agar plate inoculated from a frozen glycerol stock andused for maize transformations by methods disclosed in Example 24.

Several attempts were made to introduce DNA of plasmid pDAB101556 intostrain DAt20711 (see Example 9), a recombination-proficient ternarysystem. In all cases, plasmid pDAB101556 was found to be unstable inthis strain and a Dat20711(pDAB101556) strain was not constructed.

Plasmid pDAB101556 was successfully introduced by electroporation intocells of A. tumefaciens strain DAt16174 (Example 22) to produce strainDAt16174(pDAB101556). This strain/plasmid combination thus comprises aSUPERCHROME/binary plant transformation system. Transformants selectedby means of resistance to Streptomycin, Kanamycin, and Spectinomycinwere validated by restriction enzyme digestion of plasmid DNA prior topreparation of frozen glycerol stocks and storage at −80° C. Bulk cellsof strain DAt16174(pDAB101556) were harvested from an agar plateinoculated from a frozen glycerol stock and used for maizetransformations by methods disclosed in Example 24.

Example 24: Transformation of Maize by Agrobacterium Strains HarboringBinary Vector pDAB101556

Immature Embryo Production:

Seeds from a B104 inbred were planted into 3.5-inch SVD pots with METROMIX 360 (SUN GRO HORTICULTURE Inc.; Bellevue, Wash.). When the plantsreached the V4-V5 growth stage, they were transplanted into 4-gallonpots containing a 1:1 mix of METRO MIX 360 and PROFILE GREENS GRADEcalcined clay (PROFILE PRODUCTS LLC; Buffalo Grove, Ill.), with 20 gramsof OSMOCOTE 19-6-12, and 20 grams of IRONITE™ as additives. The plantswere grown in a greenhouse using a combination of 1000 W HPS (highpressure sodium) and 1000 W MH (metal halide) lamps set to a 16:8light/dark photoperiod if outside light did not exceed 450 W/m². Inorder to obtain immature embryos for transformation, controlled sib orself pollinations were performed.

Immature embryos were isolated at 10 to 13 days post-pollination whenembryos were approximately 1.6 to 2.0 mm in size.

Infection and Co-Cultivation:

Maize ears were surface sterilized by immersing in 20% commercial bleachwith LIQUINOX™ detergent (one or two drops per 500 mL) for 20 minutesand triple-rinsed with sterile water. A suspension of Agrobacteriumcells containing binary vector pDAB101556 was prepared from bacteriagrown on AB solid medium at 20° C. for two to three days, followed bygrowth on YEP solid medium at 28° C. for one to two days. Both the ABand YEP media contained appropriate antibiotics supplements as describedin Example 23 for each Agrobacterium strain tested with binary vectorpDAB101556. Loopfuls of cells scraped from a YEP plate were transferredinto 10 to 15 mL of liquid infection medium comprising: MS salts (Frameet al., supra), ISU Modified MS Vitamins (Frame et al., supra), 3.3 mg/LDicamba-ethanol, 68.4 gm/L sucrose, 36 gm/L glucose, 700 mg/L L-proline,pH 5.2, and containing 100-200 μM acetosyringone. The solution wasgently pipetted up and down using a sterile 5 mL pipette or vortex mixeruntil a uniform suspension was achieved, and the concentration wasadjusted to an optical density of about 1.0 at 600 nm (OD₆₀₀) using aHewlett-Packard P8452a spectrophotometer.

Co-Cultivation:

Immature embryos were isolated directly into a micro centrifuge tubecontaining 2 mL of the infection medium. The medium was removed andreplaced with 1 to 2 mL of fresh infection medium, then replaced with1.5 mL of the Agrobacterium solution. The Agrobacterium and embryosolution was incubated for 5 minutes at room temperature and thentransferred to co-cultivation medium which contained MS salts, ISUModified MS Vitamins, 3.3 mg/L Dicamba-ethanol, 30 gm/L sucrose, 700mg/L L-proline, 100 mg/L myo-inositol, 100 mg/L Casein EnzymaticHydrolysate, 15 mg/L AgNO₃, 100-200 μM acetosyringone, and 2.3 gm/LGELRITE™ (SIGMA-ALDRICH; St. Louis, Mo.), at pH 5.8. Co-cultivationincubation was for three days in the dark at 20° C.

Resting and Selection:

After co-cultivation, the embryos were transferred to a non-selectionMS-based resting medium containing MS salts, ISU Modified MS Vitamins,3.3 mg/L Dicamba-ethanol, 30 gm/L sucrose, 700 mg/L L-proline, 100 mg/Lmyo-inositol, 100 mg/L Casein Enzymatic Hydrolysate, 15 mg/L AgNO₃, 0.5gm/L MES, 250 mg/L Cefotaxime, and 2.3 gm/L GELRITE™, at pH 5.8.Incubation was continued for seven days in the dark at 28° C. Followingthe seven-day resting period, the embryos were transferred to SelectiveMedium. For selection of maize tissues transformed with a superbinary orbinary plasmid containing a plant expressible aad-1 selectable markergene, the MS-based resting medium (above) was used supplemented withHaloxyfop. The embryos were first transferred to Selection Medium Icontaining 100 nM Haloxyfop and incubated for two weeks, and thentransferred to Selection Medium II with 500 nM Haloxyfop and incubatedfor an additional two weeks. Transformed isolates were obtained over thecourse of approximately five weeks at 28° C. in the dark. If necessary,recovered isolates were bulked up by transferring to fresh SelectionMedium II for another two weeks before being transferred to regenerationmedia.

Those skilled in the art of maize transformation will understand thatother methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g., herbicidetolerance genes) are used.

Regeneration I:

Following the selection process, cultures were transferred to anMS-based Regeneration Medium I containing MS salts, ISU Modified MSVitamins, 60 gm/L sucrose, 350 mg/L L-proline, 100 mg/L myo-inositol, 50mg/L Casein Enzymatic Hydrolysate, 1 mg/L AgNO₃, 250 mg/L Cefotaxime,2.5 gm/L GELRITE™ and 500 nM Haloxyfop, at pH 5.8. Incubation wascontinued for two weeks at 28° C. in the dark.

Regeneration II:

The cultures were transferred to an MS-based Regeneration Medium IIcontaining MS salts, ISU Modified MS Vitamins, 30 gm/L sucrose, 100 mg/Lmyo-inositol, 250 mg/L Cefotaxime, 2.5 gm/L GELRITE™, and 500 nMHaloxyfop, at pH 5.8. After three weeks at 28° C. under 16/8 hoursphotoperiod, with white fluorescent light conditions (approximately 80μEm⁻² s⁻¹), plantlets were excised and transferred to an MS-based or (½MS-based) shoot/root elongation medium composed of MS salts (or ½ MSsalts), ISU Modified MS Vitamins, 0.5 gm/L MES, 30 gm/L sucrose, 100mg/L myo-inositol, 2.5 gm/L GELRITE™, at pH 5.8. When plantlets reached4 to 6 cm in length, they were transferred to the growth chamber andeventually to the greenhouse.

Seed Production:

Regenerated plants were transplanted into 3.5-inch SVD pots with METROMIX 360 and placed in a growth chamber to harden off. When plantsreached the V3 growth stage they were moved to the greenhouse, and atthe V4/V5 growth stage, they were transplanted into 5-gallon potscontaining a 1:1 mix of METRO MIX 360 and PROFILE GREENS GRADE calcinedclay, with 20 grams of OSMOCOTE 19-6-12, and 20 grams of IRONITE™ asadditives. The plants were grown in the greenhouse under a 16:8light/dark photoperiod. T1 seed was produced by performing controlledpollinations (backcross to B104). Seed was harvested six weeks afterpollination.

Multiple maize transformation experiments were performed with engineeredA. tumefaciens strains LBA4404(pDAB101556), DAt13192(pDAB101556), andDAt16174(pDAB101556), and transgenic calli selected on inhibitoryconcentrations of Haloxyfop were carried forward for plantletregeneration and further studies. In total, 16 events were retained inthe LBA4404(pDAB101556) transformations, 49 events were retained in theDAt13192(pDAB101556) transformations, and 60 events were retained in theDAt16174(pDAB101556) transformations (Table 7).

Copy numbers of the aad-1 transgene in transgenic TO plants wereestimated by hydrolysis probe assays ((Bubner and Baldwin, supra) usinggene-specific oligonucleotides. Southern blot analyses of NcoI-cleavedDNA prepared from the selected events by a cetyl trimethylammoniumbromide extraction method were performed using a PCR amplified fragmentof the aad-1 gene as 32P-labeled probe. Further, the presence ofintegrated backbone vector sequences originating from pDAB101556 wasdetected by hydrolysis probe analyses.

Thus, strain DAt16174, a SUPERCHROME strain comprising a full set ofpTiACH5-derived vir genes harbored on pAL4404, and further comprising apartial set of pTiBo542-derived vir genes integrated into the LBA4404chromosome at the nilA locus, is able to efficiently produce transformedmaize plants. Further, while having a somewhat lower overalltransformation efficiency than that obtained with the ternary strain,the quality of SUPERCHROME-produced events is superior, with 90% of theevents produced having single copy inserts with no detectable backbonecontamination.

While this invention has been described in certain example embodiments,which are intended as illustrative of a few aspects of the invention,the present invention may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the invention using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein. The references discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention.

TABLE 1 Representative incompatibility groups and some example plasmidsthat are classified as belonging to these incompatibility groups.Incompatibility Group Plasmids FI F, R386 FII R1 FIII Col B-K99, ColB-K166 FIV R124 I R62, R64, R483 (at least five subgroups) J R391 N R46O R724 P RP4, RK2 Q RSF1010 T R401 W R388, S-a

TABLE 2 Summary of in vitro bioassay results. Mean CEW Score Mean FAWScore Mean ECB % Damage Construct (96-well assay) (96-well assay)(32-well assay) Negative 1.64 1.78 75.9 Control 101513 0.59 0.88 17.4101514 0.80 0.78 12.0

TABLE 3 Production (in ppm; parts per million) of the AAD1, Cry1Ca,Cry1Fa, and Cry1Ab proteins in maize plants transformed with binaryvector pDAB101513. Event Name AAD1 Cry1Ca Cry1Fa Cry1Ab101513[37]-008.002 830 370 160 84 101513[37]-020.001 550 410 210 100101513[39]-011.002 380 270 150 27 101513[44]-031.003 740 300 100 40101513[45]-022.002 380 270 86 32 101513[45]-023.001 300 340 160 31101513[49]-040.002 220 270 170 21 101513[49]-040.003 210 410 220 26101513[49]-041.001 340 270 200 21

TABLE 4 Results of maize transformation experiments with strains of A.tumefaciens harboring plasmid pDAB101513. C F H Regenerable E Low-Copy GEvents of Col. B events (% D Events Events with Events of Col. G activeagainst A Embryos X-form. T₀ Events with all 4 all 4 genes F producingall all three pests Strain Treated efficiency) Analyzed genes (%) (%) 4proteins (%) (%) EHA105 2469  6 (0.24) 4  1 (25)  1 (25) 0 (0) 0 (0)DA2552 630 0 (0)   0 0 (0) 0 (0) 0 (0) 0 (0) DAt13192 1945 34 (1.75) 2521 (84) 14 (56)  9 (64)  9 (100)

TABLE 5 Results of maize transformation experiments with strains of A.tumefaciens harboring plasmid pDAB101514. C F Regenerable E Low-Copy G HB events (% D Events Events with Events of Col. Events of Col. G AEmbryos X-form. T₀ Events with all 4 all 4 genes F producing all activeagainst all Strain Treated efficiency) Analyzed genes (%) (%) 4 proteins(%) three pests (%) EHA105 3499 11 (0.31) 11  2 (18) 1 (50) 0 (0) 0 (0)DA2552 771 0 (0)   0 0 (0) 0 (0)  0 (0) 0 (0) DAt13192 926 17 (1.83) 1512 (80) 9 (75)  8 (89)  6 (75)

TABLE 6 PCR primers used for molecular confirmation ofintegration of the 15,549 bp fragment contain-ing the 14.8 KpnI VirBCDG fragment of pSB1 andKanamycin resistance gene into the nilA genomicregion of the Agrobacterium tumefaciens strain DAt16174 chromosome. Pri-Ampli- mer SEQ ID Primer Primer Sequence con Pair NO: Name (5′ to 3′)Size 1 SEQ ID H3-2 ATCTTACCTTCCTTTTCG 4,248 NO: 26 Down TTTTCCAAC bpSEQ ID Set2 CTGCTTGGATGCCCGAGG NO: 27 5′ CATAGAC 2 SEQ ID VirCATCCAAGCAGCAAGCGC 7,696 NO: 28 Screen GTTACG bp 1 5′ SEQ ID VirGTCTATGCCTCGGGCATC NO: 29 Screen CAAGCAG 4 3′ 3 SEQ ID VirGAGACCGTAGGTGATAAG 6,917 NO: 30 Screen TTGCCC bp 5 5′ SEQ ID VirTCTCATTTAGGGGCTGGC NO: 31 Screen TCCAAC 8 3′ 4 SEQ ID VirGTGCGAGCAACATGGTCAA 3,650 NO: 32 ACTCAG bp SEQ ID VirB GACATGCAGAACAACGAGNO: 33 1 3′ AAACGA 5 SEQ ID PSB1- GCACACCGAAATGCTTGG 4,126 NO: 34 1 5′TGTAGA bp SEQ ID nilA GGCCGTGCACGGCATCAA NO: 35 For1 TCTCGAA

TABLE 7 Analyses of transgenic events produced by three Agrobacteriumstrains harboring plasmid pDAB101556. Total % With No. With Events %With Single-Copy Single-Copy With Transformation Single-Copy Inserts,and Inserts, and Agrobacterium System Inserts Frequency (%) InsertsBackbone-Free Backbone-Free Ternary 60 8.5 33 75 15 DAt13192(pDAB101556)SUPERCHROME 49 6 43 90 19 DAt16174(pDAB101556) Binary 16 3 88 86 14LBA4404(pDAB101556)

What is claimed is:
 1. A method for transforming a plant, comprisingcontacting a cell of the plant with an Agrobacterium strain having atleast one pTi helper plasmid comprising a 14.8 KpnI fragment of pSB1 anda pTi plasmid having at least one disarmed T-DNA region, the T-DNAregion comprising at least a right T-DNA border and exogenous DNAadjacent to the border, wherein the plasmids have differing origins ofreplication relative to each other.
 2. The method of claim 1, whereinthe 14.8 KpnI VirBCDG fragment isolated from pSB1 in Agrobacteriumstrains has a deficiency in RecA function.
 3. A method for transforminga plant, comprising contacting a cell of the plant with a bacterium ofthe genus Agrobacterium having a 14.8 KpnI VirBCDG fragment of pSB1 anda pTi plasmid having at least one disarmed T-DNA region, wherein the14.8 KpnI VirBCDG fragment has been integrated into a neutralintegration site of a chromosome of the bacterium.
 4. The method fortransforming a plant according to claim 1, wherein the bacterium furthercomprises a plasmid having a T-DNA region adjacent to at least oneAgrobacterium T-DNA border, the plasmid having a replication origin ofan IncP incompatibility group.
 5. The method for transforming a plantaccording to claim 3, wherein the bacterium further comprises a plasmidhaving a T-DNA region adjacent to at least one Agrobacterium T-DNAborder.
 6. The method for transforming a plant according to claim 3,wherein the Agrobacterium strain is deficient in RecA functionality. 7.The method for transforming a plant according to claim 4, wherein theT-DNA region contains three or more gene sequences.
 8. The method fortransforming a plant according to claim 4, wherein the T-DNA regioncontains equal to or greater than 25,000 nucleotide base pairs.
 9. Themethod for transforming a plant according to claim 4, wherein the T-DNAregion is inserted into a single location in the plant cell when theplant is transformed.
 10. The method for transforming a plant accordingto claim 4, wherein the T-DNA region comprises more than one genesequence and the gene sequences have equal to or greater than 60%sequence homology.
 11. The method for transforming a plant according toclaim 4, wherein the T-DNA region encodes one or more of an insecticidalprotein, a herbicidal protein, or a mixture of insecticidal proteins andherbicide tolerance proteins.
 12. The method for transforming a plantaccording to claim 4, wherein the T-DNA region encodes a Cry1 Cainsecticidal protein, a Cry1F insecticidal protein, and a Cry1Ab1insecticidal protein.
 13. The method for transforming a plant accordingto claim 4, wherein the T-DNA region encodes a Cry1Ca insecticidalprotein, a Cry1F insecticidal protein, a Cry1Ab1 insecticidal protein,and an AAD-1 herbicide tolerance protein.
 14. The method according toclaim 1, wherein the plant is a monocot.
 15. The method according toclaim 1, wherein the 14.8 KpnI VirBCDG fragment is cloned into the Kpn Isite of a pDAB9291 plasmid.
 16. The method according to claim 1, whereinthe pTi helper plasmid is plasmid pMP90.
 17. The method according toclaim 1, wherein the pTi helper plasmid is plasmid pTiC58Δ.
 18. Themethod according to claim 15, further comprising transforming theAgrobacterium strain using plasmid pDAB9292 DNA.
 19. The methodaccording to claim 1, further comprising a step of selecting atransformed cell or a transformed tissue, after subjecting said culturedtissue to transformation.
 20. An Agrobacterium strain having at leastone pTi helper plasmid comprising a 14.8 KpnI fragment of pSB1 and a pTiplasmid having at least one disarmed T-DNA region, wherein the plasmidshave differing origins of replication relative to each other.
 21. TheAgrobacterium strain of claim 20, wherein the Agrobacterium strain has adeficiency in RecA function.
 22. An Agrobacterium strain havingtransformation-enhancing properties comprising a 14.8 KpnI VirBCDGfragment isolated from pSB1 and a pTi plasmid having at least onedisarmed T-DNA region.
 23. The Agrobacterium strain according to claim20, wherein the bacterium further comprises a plasmid having a T-DNAregion adjacent to at least one Agrobacterium T-DNA border.
 24. TheAgrobacterium strain according to claim 22, wherein the bacteriumfurther comprises a plasmid having a T-DNA region adjacent to at leastone Agrobacterium T-DNA border.
 25. The Agrobacterium strain accordingto claim 24, wherein the Agrobacterium strain is deficient in RecAfunctionality.
 26. The Agrobacterium strain according to claim 23,wherein the T-DNA region contains three or more gene sequences.
 27. TheAgrobacterium strain according to claim 23, wherein the T-DNA regioncontains equal to or greater than 25,000 nucleotides.
 28. TheAgrobacterium strain according to claim 23, wherein the T-DNA regioncomprises more than one gene sequence and the gene sequences havegreater than 60% sequence homology.
 29. The Agrobacterium strainaccording to claim 23, wherein T-DNA region encodes one or more of aninsecticidal protein, a herbicidal proteins, or a mixture ofinsecticidal proteins and herbicide tolerance proteins.
 30. TheAgrobacterium strain according to claim 23, wherein the T-DNA regionencodes a Cry1Ca insecticidal protein, a Cry1F insecticidal protein, anda Cry1Ab1 insecticidal protein.
 31. The Agrobacterium strain accordingto claim 23, wherein the T-DNA region encodes a Cry1Ca insecticidalprotein, a Cry1F insecticidal protein, a Cry1Ab1 insecticidal protein,and an AAD-1 herbicide tolerance protein.
 32. A nilA genomic locus ofAgrobacterium tumefaciens, wherein a polynucleotide sequence isintegrated into the nilA genomic locus.
 33. The nilA genomic locus ofclaim 32, wherein the polynucleotide sequence comprises a vir gene. 34.An Agrobacterium strain with a 14.8 KpnI VirBCDG fragment of SB1integrated into a neutral integration site on the Agrobacteriumchromosome.
 35. The Agrobacterium strain according to claim 34, whereinthe neutral integration site is a nilA genomic locus.
 36. TheAgrobacterium strain according to claim 34, wherein the Agrobacteriumstrain is deficient in RecA functionality.
 37. The Agrobacterium strainaccording to claim 34, wherein the Agrobacterium strain is Agrobacteriumtumefaciens.
 38. An Agrobacterium strain LB4404 comprising a 14.8 KpnIVirBCDG fragment of pSB1on a pTi helper plasmid and a pTi plasmid havingat least one disarmed T-DNA region and has exogenous DNA adjacent to atleast one Agrobacterium T-DNA border, wherein the plasmids havediffering origins of replication relative to each other.
 39. A plantaccording to claim
 1. 40. The plant according to claim 39, wherein anygenetic traits introduced to the plant by the transformation are stablyproduced in progeny of the plant.
 41. A plant according to claim 4,wherein the T-DNA region is stably incorporated into the plant DNA. 42.The plant according to claim 41, wherein any genes encoded by the T-DNAregion are expressed in the plant.
 43. The plant according to claim 41,wherein any genes encoded by the T-DNA region are stably produced inprogeny of the plant.
 44. The plant according to claim 41, wherein theplant stably expresses Cry1Ca insecticidal proteins, Cry1F insecticidalproteins, Cry1Ab1 insecticidal proteins, and AAD1 herbicidal proteins.45. The plant according to claim 44, wherein the plant is maize. 46.Agrobacterium strain LBA4404 comprising at least one vir gene from a14.8 KpnI VirBCDG fragment isolated from pSB1 integrated into a neutralintegration site on the Agrobacterium chromosome.
 47. A fertiletransgenic corn plant, or progeny thereof, which expresses insecticidalamounts of Cry1Ca protein, Cry1F insecticidal protein, Cry1Ab1insecticidal protein, and herbicide-tolerant amounts of AAD-1 protein,wherein the Cry1 Ca, Cry1F, Cry1Ab1, and AAD1 proteins are collectivelyexpressed from a single locus of recombinant DNA stably incorporated inthe genome of the plant.
 48. The fertile transgenic corn plant of claim47, wherein the single locus of recombinant DNA is substantially free ofvector backbone sequences from a pTi DNA plasmid.